strong purifying selection: Topics by Science.gov

Sample records for strong purifying selection

Pollen-specific, but not sperm-specific, genes show stronger purifying selection and higher rates of positive selection than sporophytic genes in Capsella grandiflora.

PubMed

Arunkumar, Ramesh; Josephs, Emily B; Williamson, Robert J; Wright, Stephen I

2013-11-01

Selection on the gametophyte can be a major force shaping plant genomes as 7-11% of genes are expressed only in that phase and 60% of genes are expressed in both the gametophytic and sporophytic phases. The efficacy of selection on gametophytic tissues is likely to be influenced by sexual selection acting on male and female functions of hermaphroditic plants. Moreover, the haploid nature of the gametophytic phase allows selection to be efficient in removing recessive deleterious mutations and fixing recessive beneficial mutations. To assess the importance of gametophytic selection, we compared the strength of purifying selection and extent of positive selection on gametophyte- and sporophyte-specific genes in the highly outcrossing plant Capsella grandiflora. We found that pollen-exclusive genes had a larger fraction of sites under strong purifying selection, a greater proportion of adaptive substitutions, and faster protein evolution compared with seedling-exclusive genes. In contrast, sperm cell-exclusive genes had a smaller fraction of sites under strong purifying selection, a lower proportion of adaptive substitutions, and slower protein evolution compared with seedling-exclusive genes. Observations of strong selection acting on pollen-expressed genes are likely explained by sexual selection resulting from pollen competition aided by the haploid nature of that tissue. The relaxation of selection in sperm might be due to the reduced influence of intrasexual competition, but reduced gene expression may also be playing an important role.
Somatic Mutation Patterns in Hemizygous Genomic Regions Unveil Purifying Selection during Tumor Evolution

PubMed Central

Basu, Swaraj; Larsson, Erik

2016-01-01

Identification of cancer driver genes using somatic mutation patterns indicative of positive selection has become a major goal in cancer genomics. However, cancer cells additionally depend on a large number of genes involved in basic cellular processes. While such genes should in theory be subject to strong purifying (negative) selection against damaging somatic mutations, these patterns have been elusive and purifying selection remains inadequately explored in cancer. Here, we hypothesized that purifying selection should be evident in hemizygous genomic regions, where damaging mutations cannot be compensated for by healthy alleles. Using a 7,781-sample pan-cancer dataset, we first confirmed this in POLR2A, an essential gene where hemizygous deletions are known to confer elevated sensitivity to pharmacological suppression. We next used this principle to identify several genes and pathways that show patterns indicative of purifying selection to avoid deleterious mutations. These include the POLR2A interacting protein INTS10 as well as genes involved in mRNA splicing, nonsense-mediated mRNA decay and other RNA processing pathways. Many of these genes belong to large protein complexes, and strong overlaps were observed with recent functional screens for gene essentiality in human cells. Our analysis supports that purifying selection acts to preserve the remaining function of many hemizygously deleted essential genes in tumors, indicating vulnerabilities that might be exploited by future therapeutic strategies. PMID:28027311
Strong Purifying Selection at Synonymous Sites in D. melanogaster

PubMed Central

Lawrie, David S.; Messer, Philipp W.; Hershberg, Ruth; Petrov, Dmitri A.

2013-01-01

Synonymous sites are generally assumed to be subject to weak selective constraint. For this reason, they are often neglected as a possible source of important functional variation. We use site frequency spectra from deep population sequencing data to show that, contrary to this expectation, 22% of four-fold synonymous (4D) sites in Drosophila melanogaster evolve under very strong selective constraint while few, if any, appear to be under weak constraint. Linking polymorphism with divergence data, we further find that the fraction of synonymous sites exposed to strong purifying selection is higher for those positions that show slower evolution on the Drosophila phylogeny. The function underlying the inferred strong constraint appears to be separate from splicing enhancers, nucleosome positioning, and the translational optimization generating canonical codon bias. The fraction of synonymous sites under strong constraint within a gene correlates well with gene expression, particularly in the mid-late embryo, pupae, and adult developmental stages. Genes enriched in strongly constrained synonymous sites tend to be particularly functionally important and are often involved in key developmental pathways. Given that the observed widespread constraint acting on synonymous sites is likely not limited to Drosophila, the role of synonymous sites in genetic disease and adaptation should be reevaluated. PMID:23737754
Population Level Purifying Selection and Gene Expression Shape Subgenome Evolution in Maize.

PubMed

Pophaly, Saurabh D; Tellier, Aurélien

2015-12-01

The maize ancestor experienced a recent whole-genome duplication (WGD) followed by gene erosion which generated two subgenomes, the dominant subgenome (maize1) experiencing fewer deletions than maize2. We take advantage of available extensive polymorphism and gene expression data in maize to study purifying selection and gene expression divergence between WGD retained paralog pairs. We first report a strong correlation in nucleotide diversity between duplicate pairs, except for upstream regions. We then show that maize1 genes are under stronger purifying selection than maize2. WGD retained genes have higher gene dosage and biased Gene Ontologies consistent with previous studies. The relative gene expression of paralogs across tissues demonstrates that 98% of duplicate pairs have either subfunctionalized in a tissuewise manner or have diverged consistently in their expression thereby preventing functional complementation. Tissuewise subfunctionalization seems to be a hallmark of transcription factors, whereas consistent repression occurs for macromolecular complexes. We show that dominant gene expression is a strong determinant of the strength of purifying selection, explaining the inferred stronger negative selection on maize1 genes. We propose a novel expression-based classification of duplicates which is more robust to explain observed polymorphism patterns than the subgenome location. Finally, upstream regions of repressed genes exhibit an enrichment in transposable elements which indicates a possible mechanism for expression divergence. © The Author 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.
Stabilizing Selection, Purifying Selection, and Mutational Bias in Finite Populations

PubMed Central

Charlesworth, Brian

2013-01-01

Genomic traits such as codon usage and the lengths of noncoding sequences may be subject to stabilizing selection rather than purifying selection. Mutations affecting these traits are often biased in one direction. To investigate the potential role of stabilizing selection on genomic traits, the effects of mutational bias on the equilibrium value of a trait under stabilizing selection in a finite population were investigated, using two different mutational models. Numerical results were generated using a matrix method for calculating the probability distribution of variant frequencies at sites affecting the trait, as well as by Monte Carlo simulations. Analytical approximations were also derived, which provided useful insights into the numerical results. A novel conclusion is that the scaled intensity of selection acting on individual variants is nearly independent of the effective population size over a wide range of parameter space and is strongly determined by the logarithm of the mutational bias parameter. This is true even when there is a very small departure of the mean from the optimum, as is usually the case. This implies that studies of the frequency spectra of DNA sequence variants may be unable to distinguish between stabilizing and purifying selection. A similar investigation of purifying selection against deleterious mutations was also carried out. Contrary to previous suggestions, the scaled intensity of purifying selection with synergistic fitness effects is sensitive to population size, which is inconsistent with the general lack of sensitivity of codon usage to effective population size. PMID:23709636
Massive expansion and differential evolution of small heat shock proteins with wheat (Triticum aestivum L.) polyploidization.

PubMed

Wang, Xiaoming; Wang, Ruochen; Ma, Chuang; Shi, Xue; Liu, Zhenshan; Wang, Zhonghua; Sun, Qixin; Cao, Jun; Xu, Shengbao

2017-05-31

Wheat (Triticum aestivum), one of the world's most important crops, is facing unprecedented challenges due to global warming. To evaluate the gene resources for heat adaptation in hexaploid wheat, small heat shock proteins (sHSPs), the key plant heat protection genes, were comprehensively analysed in wheat and related species. We found that the sHSPs of hexaploid wheat were massively expanded in A and B subgenomes with intrachromosomal duplications during polyploidization. These expanded sHSPs were under similar purifying selection and kept the expressional patterns with the original copies. Generally, a strong purifying selection acted on the α-crystallin domain (ACD) and theoretically constrain conserved function. Meanwhile, weaker purifying selection and strong positive selection acted on the N-terminal region, which conferred sHSP flexibility, allowing adjustments to a wider range of substrates in response to genomic and environmental changes. Notably, in CI, CV, ER, MI and MII subfamilies, gene duplications, expression variations and functional divergence occurred before wheat polyploidization. Our results indicate the massive expansion of active sHSPs in hexaploid wheat may also provide more raw materials for evolving functional novelties and generating genetic diversity to face future global climate changes, and highlight the expansion of stress response genes with wheat polyploidization.
Duplication and population dynamics shape historic patterns of selection and genetic variation at the major histocompatibility complex in rodents

PubMed Central

Winternitz, Jamie C; Wares, John P

2013-01-01

Genetic variation at the major histocompatibility complex (MHC) is vitally important for wildlife populations to respond to pathogen threats. As natural populations can fluctuate greatly in size, a key issue concerns how population cycles and bottlenecks that could reduce genetic diversity will influence MHC genes. Using 454 sequencing, we characterized genetic diversity at the DRB Class II locus in montane voles (Microtus montanus), a North American rodent that regularly undergoes high-amplitude fluctuations in population size. We tested for evidence of historic balancing selection, recombination, and gene duplication to identify mechanisms maintaining allelic diversity. Counter to our expectations, we found strong evidence of purifying selection acting on the DRB locus in montane voles. We speculate that the interplay between population fluctuations and gene duplication might be responsible for the weak evidence of historic balancing selection and strong evidence of purifying selection detected. To further explore this idea, we conducted a phylogenetically controlled comparative analysis across 16 rodent species with varying demographic histories and MHC duplication events (based on the maximum number of alleles detected per individual). On the basis of phylogenetic generalized linear model-averaging, we found evidence that the estimated number of duplicated loci was positively related to allelic diversity and, surprisingly, to the strength of purifying selection at the DRB locus. Our analyses also revealed that species that had undergone population bottlenecks had lower allelic richness than stable species. This study highlights the need to consider demographic history and genetic structure alongside patterns of natural selection to understand resulting patterns of genetic variation at the MHC. PMID:23789067
Immune Selection In Vitro Reveals Human Immunodeficiency Virus Type 1 Nef Sequence Motifs Important for Its Immune Evasion Function In Vivo

PubMed Central

Lee, Patricia; Ng, Hwee L.; Yang, Otto O.

2012-01-01

Human immunodeficiency virus type 1 (HIV-1) Nef downregulates major histocompatibility complex class I (MHC-I), impairing the clearance of infected cells by CD8+ cytotoxic T lymphocytes (CTLs). While sequence motifs mediating this function have been determined by in vitro mutagenesis studies of laboratory-adapted HIV-1 molecular clones, it is unclear whether the highly variable Nef sequences of primary isolates in vivo rely on the same sequence motifs. To address this issue, nef quasispecies from nine chronically HIV-1-infected persons were examined for sequence evolution and altered MHC-I downregulatory function under Gag-specific CTL immune pressure in vitro. This selection resulted in decreased nef diversity and strong purifying selection. Site-by-site analysis identified 13 codons undergoing purifying selection and 1 undergoing positive selection. Of the former, only 6 have been reported to have roles in Nef function, including 4 associated with MHC-I downregulation. Functional testing of naturally occurring in vivo polymorphisms at the 7 sites with no previously known functional role revealed 3 mutations (A84D, Y135F, and G140R) that ablated MHC-I downregulation and 3 (N52A, S169I, and V180E) that partially impaired MHC-I downregulation. Globally, the CTL pressure in vitro selected functional Nef from the in vivo quasispecies mixtures that predominately lacked MHC-I downregulatory function at the baseline. Overall, these data demonstrate that CTL pressure exerts a strong purifying selective pressure for MHC-I downregulation and identifies novel functional motifs present in Nef sequences in vivo. PMID:22553319
Pleistocene divergence across a mountain range and the influence of selection on mitogenome evolution in threatened Australian freshwater cod species.

PubMed

Harrisson, K; Pavlova, A; Gan, H M; Lee, Y P; Austin, C M; Sunnucks, P

2016-06-01

Climatic differences across a taxon's range may be associated with specific bioenergetic demands and may result in genetics-based metabolic adaptation, particularly in aquatic ectothermic organisms that rely on heat exchange with the environment to regulate key physiological processes. Extending down the east coast of Australia, the Great Dividing Range (GDR) has a strong influence on climate and the evolutionary history of freshwater fish species. Despite the GDR acting as a strong contemporary barrier to fish movement, many species, and species with shared ancestries, are found on both sides of the GDR, indicative of historical dispersal events. We sequenced complete mitogenomes from the four extant species of the freshwater cod genus Maccullochella, two of which occur on the semi-arid, inland side of the GDR, and two on the mesic coastal side. We constructed a dated phylogeny and explored the relative influences of purifying and positive selection in the evolution of mitogenome divergence among species. Results supported mid- to late-Pleistocene divergence of Maccullochella across the GDR (220-710 thousand years ago), bringing forward previously reported dates. Against a background of pervasive purifying selection, we detected potentially functionally relevant fixed amino acid differences across the GDR. Although many amino acid differences between inland and coastal species may have become fixed under relaxed purifying selection in coastal environments rather than positive selection, there was evidence of episodic positive selection acting on specific codons in the Mary River coastal lineage, which has consistently experienced the warmest and least extreme climate in the genus.
Pleistocene divergence across a mountain range and the influence of selection on mitogenome evolution in threatened Australian freshwater cod species

PubMed Central

Harrisson, K; Pavlova, A; Gan, H M; Lee, Y P; Austin, C M; Sunnucks, P

2016-01-01

Climatic differences across a taxon's range may be associated with specific bioenergetic demands and may result in genetics-based metabolic adaptation, particularly in aquatic ectothermic organisms that rely on heat exchange with the environment to regulate key physiological processes. Extending down the east coast of Australia, the Great Dividing Range (GDR) has a strong influence on climate and the evolutionary history of freshwater fish species. Despite the GDR acting as a strong contemporary barrier to fish movement, many species, and species with shared ancestries, are found on both sides of the GDR, indicative of historical dispersal events. We sequenced complete mitogenomes from the four extant species of the freshwater cod genus Maccullochella, two of which occur on the semi-arid, inland side of the GDR, and two on the mesic coastal side. We constructed a dated phylogeny and explored the relative influences of purifying and positive selection in the evolution of mitogenome divergence among species. Results supported mid- to late-Pleistocene divergence of Maccullochella across the GDR (220–710 thousand years ago), bringing forward previously reported dates. Against a background of pervasive purifying selection, we detected potentially functionally relevant fixed amino acid differences across the GDR. Although many amino acid differences between inland and coastal species may have become fixed under relaxed purifying selection in coastal environments rather than positive selection, there was evidence of episodic positive selection acting on specific codons in the Mary River coastal lineage, which has consistently experienced the warmest and least extreme climate in the genus. PMID:26883183
MC1R diversity in Northern Island Melanesia has not been constrained by strong purifying selection and cannot explain pigmentation phenotype variation in the region.

PubMed

Norton, Heather L; Werren, Elizabeth; Friedlaender, Jonathan

2015-10-19

Variation in human skin pigmentation evolved in response to the selective pressure of ultra-violet radiation (UVR). Selection to maintain darker skin in high UVR environments is expected to constrain pigmentation phenotype and variation in pigmentation loci. Consistent with this hypothesis, the gene MC1R exhibits reduced diversity in African populations from high UVR regions compared to low-UVR non-African populations. However, MC1R diversity in non-African populations that have evolved under high-UVR conditions is not well characterized. In order to test the hypothesis that MC1R variation has been constrained in Melanesians the coding region of the MC1R gene was sequenced in 188 individuals from Northern Island Melanesia. The role of purifying selection was assessed using a modified McDonald Kreitman's test. Pairwise FST was calculated between Melanesian populations and populations from the 1000 Genomes Project. The SNP rs2228479 was genotyped in a larger sample (n = 635) of Melanesians and tested for associations with skin and hair pigmentation. We observe three nonsynonymous and two synonymous mutations. A modified McDonald Kreitman's test failed to detect a significant signal of purifying selection. Pairwise FST values calculated between the four islands sampled here indicate little regional substructure in MC1R. When compared to African, European, East and South Asian populations, Melanesians do not exhibit reduced population divergence (measured as FST) or a high proportion of haplotype sharing with Africans, as one might expect if ancestral haplotypes were conserved across high UVR populations in and out of Africa. The only common nonsynonymous polymorphism observed, rs2228479, is not significantly associated with skin or hair pigmentation in a larger sample of Melanesians. The pattern of sequence diversity here does not support a model of strong selective constraint on MC1R in Northern Island Melanesia This absence of strong constraint, as well as the recent population history of the region, may explain the observed frequencies of the derived rs2228479 allele. These results emphasize the complex genetic architecture of pigmentation phenotypes, which are controlled by multiple, possibly interacting loci. They also highlight the role that population history can play in influencing phenotypic diversity in the absence of strong natural selection.
Inference of purifying and positive selection in three subspecies of chimpanzees (Pan troglodytes) from exome sequencing.

PubMed

Bataillon, Thomas; Duan, Jinjie; Hvilsom, Christina; Jin, Xin; Li, Yingrui; Skov, Laurits; Glemin, Sylvain; Munch, Kasper; Jiang, Tao; Qian, Yu; Hobolth, Asger; Wang, Jun; Mailund, Thomas; Siegismund, Hans R; Schierup, Mikkel H

2015-03-30

We study genome-wide nucleotide diversity in three subspecies of extant chimpanzees using exome capture. After strict filtering, Single Nucleotide Polymorphisms and indels were called and genotyped for greater than 50% of exons at a mean coverage of 35× per individual. Central chimpanzees (Pan troglodytes troglodytes) are the most polymorphic (nucleotide diversity, θw = 0.0023 per site) followed by Eastern (P. t. schweinfurthii) chimpanzees (θw = 0.0016) and Western (P. t. verus) chimpanzees (θw = 0.0008). A demographic scenario of divergence without gene flow fits the patterns of autosomal synonymous nucleotide diversity well except for a signal of recent gene flow from Western into Eastern chimpanzees. The striking contrast in X-linked versus autosomal polymorphism and divergence previously reported in Central chimpanzees is also found in Eastern and Western chimpanzees. We show that the direction of selection statistic exhibits a strong nonmonotonic relationship with the strength of purifying selection S, making it inappropriate for estimating S. We instead use counts in synonymous versus nonsynonymous frequency classes to infer the distribution of S coefficients acting on nonsynonymous mutations in each subspecies. The strength of purifying selection we infer is congruent with the differences in effective sizes of each subspecies: Central chimpanzees are undergoing the strongest purifying selection followed by Eastern and Western chimpanzees. Coding indels show stronger selection against indels changing the reading frame than observed in human populations. © The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.
Purifying Selection Maintains Dosage-Sensitive Genes during Degeneration of the Threespine Stickleback Y Chromosome

PubMed Central

White, Michael A.; Kitano, Jun; Peichel, Catherine L.

2015-01-01

Sex chromosomes are subject to unique evolutionary forces that cause suppression of recombination, leading to sequence degeneration and the formation of heteromorphic chromosome pairs (i.e., XY or ZW). Although progress has been made in characterizing the outcomes of these evolutionary processes on vertebrate sex chromosomes, it is still unclear how recombination suppression and sequence divergence typically occur and how gene dosage imbalances are resolved in the heterogametic sex. The threespine stickleback fish (Gasterosteus aculeatus) is a powerful model system to explore vertebrate sex chromosome evolution, as it possesses an XY sex chromosome pair at relatively early stages of differentiation. Using a combination of whole-genome and transcriptome sequencing, we characterized sequence evolution and gene expression across the sex chromosomes. We uncovered two distinct evolutionary strata that correspond with known structural rearrangements on the Y chromosome. In the oldest stratum, only a handful of genes remain, and these genes are under strong purifying selection. By comparing sex-linked gene expression with expression of autosomal orthologs in an outgroup, we show that dosage compensation has not evolved in threespine sticklebacks through upregulation of the X chromosome in males. Instead, in the oldest stratum, the genes that still possess a Y chromosome allele are enriched for genes predicted to be dosage sensitive in mammals and yeast. Our results suggest that dosage imbalances may have been avoided at haploinsufficient genes by retaining function of the Y chromosome allele through strong purifying selection. PMID:25818858
Purifying selection and genetic drift shaped Pleistocene evolution of the mitochondrial genome in an endangered Australian freshwater fish.

PubMed

Pavlova, A; Gan, H M; Lee, Y P; Austin, C M; Gilligan, D M; Lintermans, M; Sunnucks, P

2017-05-01

Genetic variation in mitochondrial genes could underlie metabolic adaptations because mitochondrially encoded proteins are directly involved in a pathway supplying energy to metabolism. Macquarie perch from river basins exposed to different climates differ in size and growth rate, suggesting potential presence of adaptive metabolic differences. We used complete mitochondrial genome sequences to build a phylogeny, estimate lineage divergence times and identify signatures of purifying and positive selection acting on mitochondrial genes for 25 Macquarie perch from three basins: Murray-Darling Basin (MDB), Hawkesbury-Nepean Basin (HNB) and Shoalhaven Basin (SB). Phylogenetic analysis resolved basin-level clades, supporting incipient speciation previously inferred from differentiation in allozymes, microsatellites and mitochondrial control region. The estimated time of lineage divergence suggested an early- to mid-Pleistocene split between SB and the common ancestor of HNB+MDB, followed by mid-to-late Pleistocene splitting between HNB and MDB. These divergence estimates are more recent than previous ones. Our analyses suggested that evolutionary drivers differed between inland MDB and coastal HNB. In the cooler and more climatically variable MDB, mitogenomes evolved under strong purifying selection, whereas in the warmer and more climatically stable HNB, purifying selection was relaxed. Evidence for relaxed selection in the HNB includes elevated transfer RNA and 16S ribosomal RNA polymorphism, presence of potentially mildly deleterious mutations and a codon (ATP6 113 ) displaying signatures of positive selection (ratio of nonsynonymous to synonymous substitution rates (dN/dS) >1, radical change of an amino-acid property and phylogenetic conservation across the Percichthyidae). In addition, the difference could be because of stronger genetic drift in the smaller and historically more subdivided HNB with low per-population effective population sizes.
Photovoltaic performance of TiO2 electrode adsorbed with gardenia yellow purified by nonionic polymeric sorbent in dye-sensitized solar cells.

PubMed

Kwon, Oh Oun; Kim, Eui Jin; Lee, Jae Hyeok; Kim, Tae Young; Park, Kyung Hee; Kim, Sang Yook; Suh, Hwa Jin; Lee, Hyo Jung; Lee, Jae Wook

2015-02-05

To improve the photovoltaic conversion efficiency in dye-sensitized solar cells (DSSCs), TiO2 electrode adsorbed with gardenia yellow purified by nonionic polymeric sorbent was successfully formulated on nanoporous TiO2 surface. Adsorption and desorption properties of crude gardenia yellow solution on a macroporous resin, XAD-1600, were investigated to purify gardenia yellow because of its strong adsorption and desorption abilities as well as high selectivity. To this end, adsorption equilibrium and kinetic data were measured and fitted using adsorption isotherms and kinetic models. Adsorption and desorption breakthrough curves in a column packed with XAD-1600 resin was obtained to optimize the separation process of gardenia yellow. The photovoltaic performance of the photo-electrode adsorbed with the crude and purified gardenia yellow in DSSCs was compared from current-voltage measurements. The results showed that the photovoltaic conversion efficiency was highly dependent on how to separate and purify gardenia yellow as a photosensitizer. Copyright © 2014 Elsevier B.V. All rights reserved.
Pseudomonas aeruginosa arylsulfatase: a purified enzyme for the mild hydrolysis of steroid sulfates.

PubMed

Stevenson, Bradley J; Waller, Christopher C; Ma, Paul; Li, Kunkun; Cawley, Adam T; Ollis, David L; McLeod, Malcolm D

2015-10-01

The hydrolysis of sulfate ester conjugates is frequently required prior to analysis for a range of analytical techniques including gas chromatography-mass spectrometry (GC-MS). Sulfate hydrolysis may be achieved with commercial crude arylsulfatase enzyme preparations such as that derived from Helix pomatia but these contain additional enzyme activities such as glucuronidase, oxidase, and reductase that make them unsuitable for many analytical applications. Strong acid can also be used to hydrolyze sulfate esters but this can lead to analyte degradation or increased matrix interference. In this work, the heterologously expressed and purified arylsulfatase from Pseudomonas aeruginosa is shown to promote the mild enzyme-catalyzed hydrolysis of a range of steroid sulfates. The substrate scope of this P. aeruginosa arylsulfatase hydrolysis is compared with commercial crude enzyme preparations such as that derived from H. pomatia. A detailed kinetic comparison is reported for selected examples. Hydrolysis in a urine matrix is demonstrated for dehydroepiandrosterone 3-sulfate and epiandrosterone 3-sulfate. The purified P. aeruginosa arylsulfatase contains only sulfatase activity allowing for the selective hydrolysis of sulfate esters in the presence of glucuronide conjugates as demonstrated in the short three-step chemoenzymatic synthesis of 5α-androstane-3β,17β-diol 17-glucuronide (ADG, 1) from epiandrosterone 3-sulfate. The P. aeruginosa arylsulfatase is readily expressed and purified (0.9 g per L of culture) and thus provides a new and selective method for the hydrolysis of steroid sulfate esters in analytical sample preparation. Copyright © 2015 John Wiley & Sons, Ltd.
Discovering functional DNA elements using population genomic information: a proof of concept using human mtDNA.

PubMed

Schrider, Daniel R; Kern, Andrew D

2014-06-09

Identifying the complete set of functional elements within the human genome would be a windfall for multiple areas of biological research including medicine, molecular biology, and evolution. Complete knowledge of function would aid in the prioritization of loci when searching for the genetic bases of disease or adaptive phenotypes. Because mutations that disrupt function are disfavored by natural selection, purifying selection leaves a detectable signature within functional elements; accordingly, this signal has been exploited for over a decade through the use of genomic comparisons of distantly related species. While this is so, the functional complement of the genome changes extensively across time and between lineages; therefore, evidence of the current action of purifying selection in humans is essential. Because the removal of deleterious mutations by natural selection also reduces within-species genetic diversity within functional loci, dense population genetic data have the potential to reveal genomic elements that are currently functional. Here, we assess the potential of this approach by examining an ultradeep sample of human mitochondrial genomes (n = 16,411). We show that the high density of polymorphism in this data set precisely delineates regions experiencing purifying selection. Furthermore, we show that the number of segregating alleles at a site is strongly correlated with its divergence across species after accounting for known mutational biases in human mitochondrial DNA (ρ = 0.51; P < 2.2 × 10(-16)). These two measures track one another at a remarkably fine scale across many loci-a correlation that is purely the result of natural selection. Our results demonstrate that genetic variation has the potential to reveal with surprising precision which regions in the genome are currently performing important functions and likely to have deleterious fitness effects when mutated. As more complete human genomes are sequenced, similar power to reveal purifying selection may be achievable in the human nuclear genome. © The Author(s) 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.
Positive selection in the SLC11A1 gene in the family Equidae.

PubMed

Bayerova, Zuzana; Janova, Eva; Matiasovic, Jan; Orlando, Ludovic; Horin, Petr

2016-05-01

Immunity-related genes are a suitable model for studying effects of selection at the genomic level. Some of them are highly conserved due to functional constraints and purifying selection, while others are variable and change quickly to cope with the variation of pathogens. The SLC11A1 gene encodes a transporter protein mediating antimicrobial activity of macrophages. Little is known about the patterns of selection shaping this gene during evolution. Although it is a typical evolutionarily conserved gene, functionally important polymorphisms associated with various diseases were identified in humans and other species. We analyzed the genomic organization, genetic variation, and evolution of the SLC11A1 gene in the family Equidae to identify patterns of selection within this important gene. Nucleotide SLC11A1 sequences were shown to be highly conserved in ten equid species, with more than 97 % sequence identity across the family. Single nucleotide polymorphisms (SNPs) were found in the coding and noncoding regions of the gene. Seven codon sites were identified to be under strong purifying selection. Codons located in three regions, including the glycosylated extracellular loop, were shown to be under diversifying selection. A 3-bp indel resulting in a deletion of the amino acid 321 in the predicted protein was observed in all horses, while it has been maintained in all other equid species. This codon comprised in an N-glycosylation site was found to be under positive selection. Interspecific variation in the presence of predicted N-glycosylation sites was observed.
Relating surfactant properties to activity and solubilization of the human adenosine a3 receptor.

PubMed

Berger, Bryan W; García, Roxana Y; Lenhoff, Abraham M; Kaler, Eric W; Robinson, Clifford R

2005-07-01

The effects of various surfactants on the activity and stability of the human adenosine A3 receptor (A3) were investigated. The receptor was expressed using stably transfected HEK293 cells at a concentration of 44 pmol functional receptor per milligram membrane protein and purified using over 50 different nonionic surfactants. A strong correlation was observed between a surfactant's ability to remove A3 from the membrane and the ability of the surfactant to remove A3 selectively relative to other membrane proteins. The activity of A3 once purified also correlates well with the selectivity of the surfactant used. The effects of varying the surfactant were much stronger than those achieved by including A3 ligands in the purification scheme. Notably, all surfactants that gave high efficiency, selectivity and activity fall within a narrow range of hydrophile-lipophile balance values. This effect may reflect the ability of the surfactant to pack effectively at the hydrophobic transmembrane interface. These findings emphasize the importance of identifying appropriate surfactants for a particular membrane protein, and offer promise for the development of rapid, efficient, and systematic methods to facilitate membrane protein purification.
High level of microsynteny and purifying selection affect the evolution of WRKY family in Gramineae.

PubMed

Jin, Jing; Kong, Jingjing; Qiu, Jianle; Zhu, Huasheng; Peng, Yuancheng; Jiang, Haiyang

2016-01-01

The WRKY gene family, which encodes proteins in the regulation processes of diverse developmental stages, is one of the largest families of transcription factors in higher plants. In this study, by searching for interspecies gene colinearity (microsynteny) and dating the age distributions of duplicated genes, we found 35 chromosomal segments of subgroup I genes of WRKY family (WRKY I) in four Gramineae species (Brachypodium, rice, sorghum, and maize) formed eight orthologous groups. After a stepwise gene-by-gene reciprocal comparison of all the protein sequences in the WRKY I gene flanking areas, highly conserved regions of microsynteny were found in the four Gramineae species. Most gene pairs showed conserved orientation within syntenic genome regions. Furthermore, tandem duplication events played the leading role in gene expansion. Eventually, environmental selection pressure analysis indicated strong purifying selection for the WRKY I genes in Gramineae, which may have been followed by gene loss and rearrangement. The results presented in this study provide basic information of Gramineae WRKY I genes and form the foundation for future functional studies of these genes. High level of microsynteny in the four grass species provides further evidence that a large-scale genome duplication event predated speciation.

Strong signatures of selection in the domestic pig genome.

PubMed

Rubin, Carl-Johan; Megens, Hendrik-Jan; Martinez Barrio, Alvaro; Maqbool, Khurram; Sayyab, Shumaila; Schwochow, Doreen; Wang, Chao; Carlborg, Örjan; Jern, Patric; Jørgensen, Claus B; Archibald, Alan L; Fredholm, Merete; Groenen, Martien A M; Andersson, Leif

2012-11-27

Domestication of wild boar (Sus scrofa) and subsequent selection have resulted in dramatic phenotypic changes in domestic pigs for a number of traits, including behavior, body composition, reproduction, and coat color. Here we have used whole-genome resequencing to reveal some of the loci that underlie phenotypic evolution in European domestic pigs. Selective sweep analyses revealed strong signatures of selection at three loci harboring quantitative trait loci that explain a considerable part of one of the most characteristic morphological changes in the domestic pig--the elongation of the back and an increased number of vertebrae. The three loci were associated with the NR6A1, PLAG1, and LCORL genes. The latter two have repeatedly been associated with loci controlling stature in other domestic animals and in humans. Most European domestic pigs are homozygous for the same haplotype at these three loci. We found an excess of derived nonsynonymous substitutions in domestic pigs, most likely reflecting both positive selection and relaxed purifying selection after domestication. Our analysis of structural variation revealed four duplications at the KIT locus that were exclusively present in white or white-spotted pigs, carrying the Dominant white, Patch, or Belt alleles. This discovery illustrates how structural changes have contributed to rapid phenotypic evolution in domestic animals and how alleles in domestic animals may evolve by the accumulation of multiple causative mutations as a response to strong directional selection.
Strong signatures of selection in the domestic pig genome

PubMed Central

Rubin, Carl-Johan; Megens, Hendrik-Jan; Barrio, Alvaro Martinez; Maqbool, Khurram; Sayyab, Shumaila; Schwochow, Doreen; Wang, Chao; Carlborg, Örjan; Jern, Patric; Jørgensen, Claus B.; Archibald, Alan L.; Fredholm, Merete; Groenen, Martien A. M.; Andersson, Leif

2012-01-01

Domestication of wild boar (Sus scrofa) and subsequent selection have resulted in dramatic phenotypic changes in domestic pigs for a number of traits, including behavior, body composition, reproduction, and coat color. Here we have used whole-genome resequencing to reveal some of the loci that underlie phenotypic evolution in European domestic pigs. Selective sweep analyses revealed strong signatures of selection at three loci harboring quantitative trait loci that explain a considerable part of one of the most characteristic morphological changes in the domestic pig—the elongation of the back and an increased number of vertebrae. The three loci were associated with the NR6A1, PLAG1, and LCORL genes. The latter two have repeatedly been associated with loci controlling stature in other domestic animals and in humans. Most European domestic pigs are homozygous for the same haplotype at these three loci. We found an excess of derived nonsynonymous substitutions in domestic pigs, most likely reflecting both positive selection and relaxed purifying selection after domestication. Our analysis of structural variation revealed four duplications at the KIT locus that were exclusively present in white or white-spotted pigs, carrying the Dominant white, Patch, or Belt alleles. This discovery illustrates how structural changes have contributed to rapid phenotypic evolution in domestic animals and how alleles in domestic animals may evolve by the accumulation of multiple causative mutations as a response to strong directional selection. PMID:23151514
Preparative isolation and purification of seven main antioxidants from Eucommia ulmoides Oliv. (Du-zhong) leaves using HSCCC guided by DPPH-HPLC experiment.

PubMed

Dai, Xingping; Huang, Qiong; Zhou, Boting; Gong, Zhicheng; Liu, Zhaoqian; Shi, Shuyun

2013-08-15

Seven antioxidants were purified from Eucommia ulmoides Oliv. leaves using HSCCC guided by DPPH-HPLC experiment. HSCCC was successfully used to separate target antioxidants by three runs with different solvent systems after D101 column chromatography fractionation. Ethyl acetate-n-butanol-water (1:2:3, v/v/v) was selected as the optimum solvent system to purify geniposidic acid. Ethyl acetate-ethanol-water (4:1:5, v/v/v) was used to isolate caffeic acid, chlorogenic acid and ferulic acid. While three flavonoids, quercetin-3-O-sambubioside, rutin and isoquercitrin were purified by petroleum ether-ethyl acetate-methanol-water (1:5:1:5, v/v/v/v). The structures were identified by MS and NMR. Antioxidant activities were assessed, and compounds 2-7 showed strong antioxidant activities. This is the first report about separation of antioxidants from E. ulmoides leaves by HSCCC. The results indicated that the combinative methods using DPPH-HPLC and HSCCC could be widely applied for screening and isolation of antioxidants from complex extracts. Copyright © 2013 Elsevier Ltd. All rights reserved.
Evolutionary dynamics of Rh2 opsins in birds demonstrate an episode of accelerated evolution in the New World warblers (Setophaga)

PubMed Central

Price, Trevor D.

2015-01-01

Low rates of sequence evolution associated with purifying selection can be interrupted by episodic changes in selective regimes. Visual pigments are a unique system in which we can investigate the functional consequences of genetic changes, therefore connecting genotype to phenotype in the context of natural and sexual selection pressures. We study the RH2 and RH1 visual pigments (opsins) across 22 bird species belonging to two ecologically convergent clades, the New World warblers (Parulidae) and Old World warblers (Phylloscopidae), and evaluate rates of evolution in these clades along with data from 21 additional species. We demonstrate generally slow evolution of these opsins: both Rh1 and Rh2 are highly conserved across Old World and New World warblers. However, Rh2 underwent a burst of evolution within the New World genus Setophaga, where it accumulated substitutions at 6 amino acid sites across the species we studied. Evolutionary analyses revealed a significant increase in dN/dS in Setophaga, implying relatively strong selective pressures to overcome long-standing purifying selection. We studied the effects of each substitution on spectral tuning and found they do not cause large spectral shifts. Thus substitutions may reflect other aspects of opsin function, such as those affecting photosensitivity and/or dark-light adaptation. Although it is unclear what these alterations mean for color perception, we suggest that rapid evolution is linked to sexual selection, given the exceptional plumage colour diversification in Setophaga. PMID:25827331
Phylogeographic distribution of very virulent infectious bursal disease virus isolates in the Iberian Peninsula.

PubMed

Cortey, Martí; Bertran, Kateri; Toskano, Jennifer; Majó, Natàlia; Dolz, Roser

2012-01-01

Viral population dynamics of very virulent infectious bursal disease virus (vvIBDV) field strains isolated in the Iberian Peninsula since the first outbreak in the 1990s have been analysed. Low levels of genetic variability and a global purification selection pattern were reported in 480 base pairs of the hypervariable region of the VP2 gene, indicating a lack of a selection-driven immune escape in the evolutive pathway of the virus. The viral population structure of vvIBDV strains in the Iberian Peninsula showed a strong relationship between geography and phylogeny, with two main groups observed. A global comparison among vvIBDV strains also showed an association with sequences from the same country. The low variability, the strong purifying selection and the geographical pattern observed point to a picture where the virus evolves slowly, occupying the same geographical niche for a long time. The scenario depicted fits well with the biological features of the virus: being able to remain viable for long periods of time due to a strong environmental resistance, and as an immunosuppressive agent, capable per se of annihilating temporally the immune system of the host.
Theory of prokaryotic genome evolution.

PubMed

Sela, Itamar; Wolf, Yuri I; Koonin, Eugene V

2016-10-11

Bacteria and archaea typically possess small genomes that are tightly packed with protein-coding genes. The compactness of prokaryotic genomes is commonly perceived as evidence of adaptive genome streamlining caused by strong purifying selection in large microbial populations. In such populations, even the small cost incurred by nonfunctional DNA because of extra energy and time expenditure is thought to be sufficient for this extra genetic material to be eliminated by selection. However, contrary to the predictions of this model, there exists a consistent, positive correlation between the strength of selection at the protein sequence level, measured as the ratio of nonsynonymous to synonymous substitution rates, and microbial genome size. Here, by fitting the genome size distributions in multiple groups of prokaryotes to predictions of mathematical models of population evolution, we show that only models in which acquisition of additional genes is, on average, slightly beneficial yield a good fit to genomic data. These results suggest that the number of genes in prokaryotic genomes reflects the equilibrium between the benefit of additional genes that diminishes as the genome grows and deletion bias (i.e., the rate of deletion of genetic material being slightly greater than the rate of acquisition). Thus, new genes acquired by microbial genomes, on average, appear to be adaptive. The tight spacing of protein-coding genes likely results from a combination of the deletion bias and purifying selection that efficiently eliminates nonfunctional, noncoding sequences.
Sequence polymorphism in an insect RNA virus field population: A snapshot from a single point in space and time reveals stochastic differences among and within individual hosts

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stenger, Drake C., E-mail: drake.stenger@ars.usda.

Population structure of Homalodisca coagulata Virus-1 (HoCV-1) among and within field-collected insects sampled from a single point in space and time was examined. Polymorphism in complete consensus sequences among single-insect isolates was dominated by synonymous substitutions. The mutant spectrum of the C2 helicase region within each single-insect isolate was unique and dominated by nonsynonymous singletons. Bootstrapping was used to correct the within-isolate nonsynonymous:synonymous arithmetic ratio (N:S) for RT-PCR error, yielding an N:S value ~one log-unit greater than that of consensus sequences. Probability of all possible single-base substitutions for the C2 region predicted N:S values within 95% confidence limits of themore » corrected within-isolate N:S when the only constraint imposed was viral polymerase error bias for transitions over transversions. These results indicate that bottlenecks coupled with strong negative/purifying selection drive consensus sequences toward neutral sequence space, and that most polymorphism within single-insect isolates is composed of newly-minted mutations sampled prior to selection. -- Highlights: •Sampling protocol minimized differential selection/history among isolates. •Polymorphism among consensus sequences dominated by negative/purifying selection. •Within-isolate N:S ratio corrected for RT-PCR error by bootstrapping. •Within-isolate mutant spectrum dominated by new mutations yet to undergo selection.« less
Molecular organization and phylogenetic analysis of 5S rDNA in crustaceans of the genus Pollicipes reveal birth-and-death evolution and strong purifying selection.

PubMed

Perina, Alejandra; Seoane, David; González-Tizón, Ana M; Rodríguez-Fariña, Fernanda; Martínez-Lage, Andrés

2011-10-17

The 5S ribosomal DNA (5S rDNA) is organized in tandem arrays with repeat units that consist of a transcribing region (5S) and a variable nontranscribed spacer (NTS), in higher eukaryotes. Until recently the 5S rDNA was thought to be subject to concerted evolution, however, in several taxa, sequence divergence levels between the 5S and the NTS were found higher than expected under this model. So, many studies have shown that birth-and-death processes and selection can drive the evolution of 5S rDNA. In analyses of 5S rDNA evolution is found several 5S rDNA types in the genome, with low levels of nucleotide variation in the 5S and a spacer region highly divergent. Molecular organization and nucleotide sequence of the 5S ribosomal DNA multigene family (5S rDNA) were investigated in three Pollicipes species in an evolutionary context. The nucleotide sequence variation revealed that several 5S rDNA variants occur in Pollicipes genomes. They are clustered in up to seven different types based on differences in their nontranscribed spacers (NTS). Five different units of 5S rDNA were characterized in P. pollicipes and two different units in P. elegans and P. polymerus. Analysis of these sequences showed that identical types were shared among species and that two pseudogenes were present. We predicted the secondary structure and characterized the upstream and downstream conserved elements. Phylogenetic analysis showed an among-species clustering pattern of 5S rDNA types. These results suggest that the evolution of Pollicipes 5S rDNA is driven by birth-and-death processes with strong purifying selection.
Molecular organization and phylogenetic analysis of 5S rDNA in crustaceans of the genus Pollicipes reveal birth-and-death evolution and strong purifying selection

PubMed Central

2011-01-01

Background The 5S ribosomal DNA (5S rDNA) is organized in tandem arrays with repeat units that consist of a transcribing region (5S) and a variable nontranscribed spacer (NTS), in higher eukaryotes. Until recently the 5S rDNA was thought to be subject to concerted evolution, however, in several taxa, sequence divergence levels between the 5S and the NTS were found higher than expected under this model. So, many studies have shown that birth-and-death processes and selection can drive the evolution of 5S rDNA. In analyses of 5S rDNA evolution is found several 5S rDNA types in the genome, with low levels of nucleotide variation in the 5S and a spacer region highly divergent. Molecular organization and nucleotide sequence of the 5S ribosomal DNA multigene family (5S rDNA) were investigated in three Pollicipes species in an evolutionary context. Results The nucleotide sequence variation revealed that several 5S rDNA variants occur in Pollicipes genomes. They are clustered in up to seven different types based on differences in their nontranscribed spacers (NTS). Five different units of 5S rDNA were characterized in P. pollicipes and two different units in P. elegans and P. polymerus. Analysis of these sequences showed that identical types were shared among species and that two pseudogenes were present. We predicted the secondary structure and characterized the upstream and downstream conserved elements. Phylogenetic analysis showed an among-species clustering pattern of 5S rDNA types. Conclusions These results suggest that the evolution of Pollicipes 5S rDNA is driven by birth-and-death processes with strong purifying selection. PMID:22004418
Evolution of CCL11: genetic characterization in lagomorphs and evidence of positive and purifying selection in mammals.

PubMed

Neves, Fabiana; Abrantes, Joana; Esteves, Pedro J

2016-07-01

The interactions between chemokines and their receptors are crucial for differentiation and activation of inflammatory cells. CC chemokine ligand 11 (CCL11) binds to CCR3 and to CCR5 that in leporids underwent gene conversion with CCR2. Here, we genetically characterized CCL11 in lagomorphs (leporids and pikas). All lagomorphs have a potentially functional CCL11, and the Pygmy rabbit has a mutation in the stop codon that leads to a longer protein. Other mammals also have mutations at the stop codon that result in proteins with different lengths. By employing maximum likelihood methods, we observed that, in mammals, CCL11 exhibits both signatures of purifying and positive selection. Signatures of purifying selection were detected in sites important for receptor binding and activation. Of the three sites detected as under positive selection, two were located close to the stop codon. Our results suggest that CCL11 is functional in all lagomorphs, and that the signatures of purifying and positive selection in mammalian CCL11 probably reflect the protein's biological roles. © The Author(s) 2016.
Purification of carbon nanotubes via selective heating

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rogers, John A.; Wilson, William L.; Jin, Sung Hun

The present invention provides methods for purifying a layer of carbon nanotubes comprising providing a precursor layer of substantially aligned carbon nanotubes supported by a substrate, wherein the precursor layer comprises a mixture of first carbon nanotubes and second carbon nanotubes; selectively heating the first carbon nanotubes; and separating the first carbon nanotubes from the second carbon nanotubes, thereby generating a purified layer of carbon nanotubes. Devices benefiting from enhanced electrical properties enabled by the purified layer of carbon nanotubes are also described.
Evolution of the human immunodeficiency virus envelope gene is dominated by purifying selection.

PubMed

Edwards, C T T; Holmes, E C; Pybus, O G; Wilson, D J; Viscidi, R P; Abrams, E J; Phillips, R E; Drummond, A J

2006-11-01

The evolution of the human immunodeficiency virus (HIV-1) during chronic infection involves the rapid, continuous turnover of genetic diversity. However, the role of natural selection, relative to random genetic drift, in governing this process is unclear. We tested a stochastic model of genetic drift using partial envelope sequences sampled longitudinally in 28 infected children. In each case the Bayesian posterior (empirical) distribution of coalescent genealogies was estimated using Markov chain Monte Carlo methods. Posterior predictive simulation was then used to generate a null distribution of genealogies assuming neutrality, with the null and empirical distributions compared using four genealogy-based summary statistics sensitive to nonneutral evolution. Because both null and empirical distributions were generated within a coalescent framework, we were able to explicitly account for the confounding influence of demography. From the distribution of corrected P-values across patients, we conclude that empirical genealogies are more asymmetric than expected if evolution is driven by mutation and genetic drift only, with an excess of low-frequency polymorphisms in the population. This indicates that although drift may still play an important role, natural selection has a strong influence on the evolution of HIV-1 envelope. A negative relationship between effective population size and substitution rate indicates that as the efficacy of selection increases, a smaller proportion of mutations approach fixation in the population. This suggests the presence of deleterious mutations. We therefore conclude that intrahost HIV-1 evolution in envelope is dominated by purifying selection against low-frequency deleterious mutations that do not reach fixation.
[Identification and production of monoclonal antibody of Siberian tiger's immunoglobulin].

PubMed

Zhang, Yaonglong; Zhang, Duanling; Zhou, Ming; Xue, Yuan; Hua, Yuping; Ma, Jianzhang

2010-03-01

To purify immunoglobulin (Ig) of Siberian Tiger and prepare monoclonal antibody (mAb) against the Ig,which can be used to develop immunological diagnostic kits for diagnosing infectious disease in Siberian Tiger. The Ig of Siberian tigers was purified with saturated ammonium sulfate combined with recombinant Protein G. The C57BL/6 mice were immunized with the purified Ig. Spleno-cytes of the mice immunized were collected and fused with the mouse myeloma cell line (Sp2/0-Ag14). The positive hybridoma clones were selected by ELISA and were identified by western blot. The sandwich ELISA was used to detect immunocompetence of the purified Ig and the mAb. We obtained three mouse hybridoma clones that produced mAbs against Ig of Siberian Tiger. The derived McAbs could recognize Ig heavy chain of Siberian Tiger specifically. The biological activity of the Ig and obtained McAbs also could be identified by detecting the antibody induced by panleukopenia virus (FPV-HLJ) vaccine in Siberian Tiger. The antibody also would be useful for assess the vaccine efficacy against the infectious disease on the Siberian Tiger. Protein G can be used in Ig purification of Siberian Tiger. The obtained McAbs from the hybridoma ADT11 in this study owned strong ability to bind Ig of Siberian Tiger and have a stable immunocompetence. They can be used to develop diagnostic methods for detecting infectious disease in Siberian Tiger and vaccine research.
Development of Cell-SELEX Technology and Its Application in Cancer Diagnosis and Therapy.

PubMed

Chen, Man; Yu, Yuanyuan; Jiang, Feng; Zhou, Junwei; Li, Yongshu; Liang, Chao; Dang, Lei; Lu, Aiping; Zhang, Ge

2016-12-10

SELEX (systematic evolution of ligands by exponential enrichment) is a process involving the progressive isolation of high selective ssDNA/RNA from a combinatorial single-stranded oligonucleotide library through repeated rounds of binding, partitioning and amplification. SELEX-derived single-stranded DNA/RNA molecules, called aptamers, are selected against a wide range of targets, including purified proteins, live cells, tissues, microorganisms, small molecules and so on. With the development of SELEX technology over the last two decades, various modified SELEX processes have been arisen. A majority of aptamers are selected against purified proteins through traditional SELEX. Unfortunately, more and more evidence showed aptamers selected against purified membrane proteins failed to recognize their targets in live cells. Cell-SELEX could develop aptamers against a particular target cell line to discriminate this cell line from others. Therefore, cell-SELEX has been widely used to select aptamers for the application of both diagnosis and therapy of various diseases, especially for cancer. In this review, the advantages and limitations of cell-SELEX and SELEX against purified protein will be compared. Various modified cell-SELEX techniques will be summarized, and application of cell-SELEX in cancer diagnosis and therapy will be discussed.
Development of Cell-SELEX Technology and Its Application in Cancer Diagnosis and Therapy

PubMed Central

Chen, Man; Yu, Yuanyuan; Jiang, Feng; Zhou, Junwei; Li, Yongshu; Liang, Chao; Dang, Lei; Lu, Aiping; Zhang, Ge

2016-01-01

SELEX (systematic evolution of ligands by exponential enrichment) is a process involving the progressive isolation of high selective ssDNA/RNA from a combinatorial single-stranded oligonucleotide library through repeated rounds of binding, partitioning and amplification. SELEX-derived single-stranded DNA/RNA molecules, called aptamers, are selected against a wide range of targets, including purified proteins, live cells, tissues, microorganisms, small molecules and so on. With the development of SELEX technology over the last two decades, various modified SELEX processes have been arisen. A majority of aptamers are selected against purified proteins through traditional SELEX. Unfortunately, more and more evidence showed aptamers selected against purified membrane proteins failed to recognize their targets in live cells. Cell-SELEX could develop aptamers against a particular target cell line to discriminate this cell line from others. Therefore, cell-SELEX has been widely used to select aptamers for the application of both diagnosis and therapy of various diseases, especially for cancer. In this review, the advantages and limitations of cell-SELEX and SELEX against purified protein will be compared. Various modified cell-SELEX techniques will be summarized, and application of cell-SELEX in cancer diagnosis and therapy will be discussed. PMID:27973403
Cloning, expression, and purification of the virulence-associated protein D from Xylella fastidiosa.

PubMed

Catani, Cleide Ferreira; Azzoni, Adriano Rodrigues; Paula, Débora Pires; Tada, Susely Ferraz Siqueira; Rosselli, Luciana Kauer; de Souza, Anete Pereira; Yano, Tomomasa

2004-10-01

In this study, an efficient expression system, based on the pET32Xa/LIC vector, for producing a Xylella fastidiosa virulence-associated protein D, found to have a strong similarity to Riemerella anatipestifer and Actinobacillus actinomycetencomitans VapD protein, is presented. The protein has a molecular mass of 17.637 Da and a calculated pI of 5.49. The selected XFa0052 gene was cloned in the pET32Xa/LIC vector and the plasmid was transformed into Escherichia coli BL21 (DE3) strain at 37 degrees C, with an induction time of 2 h and 1 mM IPTG concentration. The protein present in the soluble fraction was purified by immobilized metal affinity chromatography (IMAC), and had its identity determined by mass spectrometry (MALDI-TOF) and N-terminal sequencing. The purified protein was found as a single band on SDS-PAGE and its correct folding was verified by circular dichroism spectroscopy.
The Role of Small RNA-Based Epigenetic Silencing for Purifying Selection on Transposable Elements in Capsella grandiflora

PubMed Central

Horvath, Robert

2017-01-01

Abstract To avoid negative effects of transposable element (TE) proliferation, plants epigenetically silence TEs using a number of mechanisms, including RNA-directed DNA methylation. These epigenetic modifications can extend outside the boundaries of TE insertions and lead to silencing of nearby genes, resulting in a trade-off between TE silencing and interference with nearby gene regulation. Therefore, purifying selection is expected to remove silenced TE insertions near genes more efficiently and prevent their accumulation within a population. To explore how effects of TE silencing on gene regulation shapes purifying selection on TEs, we analyzed whole genome sequencing data from 166 individuals of a large population of the outcrossing species Capsella grandiflora. We found that most TEs are rare, and in chromosome arms, silenced TEs are exposed to stronger purifying selection than those that are not silenced by 24-nucleotide small RNAs, especially with increasing proximity to genes. An age-of-allele test of neutrality on a subset of TEs supports our inference of purifying selection on silenced TEs, suggesting that our results are robust to varying transposition rates. Our results provide new insights into the processes affecting the accumulation of TEs in an outcrossing species and support the view that epigenetic silencing of TEs results in a trade-off between preventing TE proliferation and interference with nearby gene regulation. We also suggest that in the centromeric and pericentromeric regions, the negative aspects of epigenetic TE silencing are missing. PMID:29036316
Frequent associations between CTL and T-Helper epitopes in HIV-1 genomes and implications for multi-epitope vaccine designs

PubMed Central

2010-01-01

Background Epitope vaccines have been suggested as a strategy to counteract viral escape and development of drug resistance. Multiple studies have shown that Cytotoxic T-Lymphocyte (CTL) and T-Helper (Th) epitopes can generate strong immune responses in Human Immunodeficiency Virus (HIV-1). However, not much is known about the relationship among different types of HIV epitopes, particularly those epitopes that can be considered potential candidates for inclusion in the multi-epitope vaccines. Results In this study we used association rule mining to examine relationship between different types of epitopes (CTL, Th and antibody epitopes) from nine protein-coding HIV-1 genes to identify strong associations as potent multi-epitope vaccine candidates. Our results revealed 137 association rules that were consistently present in the majority of reference and non-reference HIV-1 genomes and included epitopes of two different types (CTL and Th) from three different genes (Gag, Pol and Nef). These rules involved 14 non-overlapping epitope regions that frequently co-occurred despite high mutation and recombination rates, including in genomes of circulating recombinant forms. These epitope regions were also highly conserved at both the amino acid and nucleotide levels indicating strong purifying selection driven by functional and/or structural constraints and hence, the diminished likelihood of successful escape mutations. Conclusions Our results provide a comprehensive systematic survey of CTL, Th and Ab epitopes that are both highly conserved and co-occur together among all subtypes of HIV-1, including circulating recombinant forms. Several co-occurring epitope combinations were identified as potent candidates for inclusion in multi-epitope vaccines, including epitopes that are immuno-responsive to different arms of the host immune machinery and can enable stronger and more efficient immune responses, similar to responses achieved with adjuvant therapies. Signature of strong purifying selection acting at the nucleotide level of the associated epitopes indicates that these regions are functionally critical, although the exact reasons behind such sequence conservation remain to be elucidated. PMID:20696039
The alpha glycerophosphate cycle in Drosophila melanogaster VI. structure and evolution of enzyme paralogs in the genus Drosophila.

PubMed

Carmon, Amber; MacIntyre, Ross

2010-01-01

The genome sequences of 12 Drosophila species contain 3 paralogs for alpha glycerophosphate dehydrogenase (GPDH) and for the mitochondrial alpha glycerophosphate oxidase (GPO). These 2 enzymes participate in the alpha glycerophosphate cycle in the adult thoracic flight muscles. The flight muscle enzymes are encoded by gpdh-1 at 26A2 and gpo-1 at 52C8. In this paper, we show that the GPDH paralogs share the same evolutionarily conserved functional domains and most intron positions, whereas the GPO paralogs share only some of the functional domains of mitochondrial oxidoreductases. The GPO paralogs not expressed in the flight muscles essentially lack introns. GPDH paralogs encoded by gpdh-2 and gpdh-3 and the GPO paralogs encoded by gpo-2 and gpo-3 are expressed only in the testes. Gene trees for the GPDH and GPO paralogs indicate that the genes expressed in the flight muscles are evolving very slowly presumably under strong purifying selection whereas the paralogs expressed in the testes are evolving more rapidly. The concordance between species and gene trees, d(N)/d(S) ratios, phylogenetic analysis by maximum likelihood-based tests, and analyses of radical and conservative substitutions all indicate that the additional GPDH and GPO paralogs are also evolving under purifying selection.
Chance and necessity in the genome evolution of endosymbiotic bacteria of insects.

PubMed

Sabater-Muñoz, Beatriz; Toft, Christina; Alvarez-Ponce, David; Fares, Mario A

2017-06-01

An open question in evolutionary biology is how does the selection-drift balance determine the fates of biological interactions. We searched for signatures of selection and drift in genomes of five endosymbiotic bacterial groups known to evolve under strong genetic drift. Although most genes in endosymbiotic bacteria showed evidence of relaxed purifying selection, many genes in these bacteria exhibited stronger selective constraints than their orthologs in free-living bacterial relatives. Remarkably, most of these highly constrained genes had no role in the host-symbiont interactions but were involved in either buffering the deleterious consequences of drift or other host-unrelated functions, suggesting that they have either acquired new roles or their role became more central in endosymbiotic bacteria. Experimental evolution of Escherichia coli under strong genetic drift revealed remarkable similarities in the mutational spectrum, genome reduction patterns and gene losses to endosymbiotic bacteria of insects. Interestingly, the transcriptome of the experimentally evolved lines showed a generalized deregulation of the genome that affected genes encoding proteins involved in mutational buffering, regulation and amino acid biosynthesis, patterns identical to those found in endosymbiotic bacteria. Our results indicate that drift has shaped endosymbiotic associations through a change in the functional landscape of bacterial genes and that the host had only a small role in such a shift.

Topology of evolving, mutagenized viral populations: quasispecies expansion, compression, and operation of negative selection.

PubMed

Ojosnegros, Samuel; Agudo, Rubén; Sierra, Macarena; Briones, Carlos; Sierra, Saleta; González-López, Claudia; Domingo, Esteban; Cristina, Juan

2008-07-17

The molecular events and evolutionary forces underlying lethal mutagenesis of virus (or virus extinction through an excess of mutations) are not well understood. Here we apply for the first time phylogenetic methods and Partition Analysis of Quasispecies (PAQ) to monitor genetic distances and intra-population structures of mutant spectra of foot-and-mouth disease virus (FMDV) quasispecies subjected to mutagenesis by base and nucleoside analogues. Phylogenetic and PAQ analyses have revealed a highly dynamic variation of intrapopulation diversity of FMDV quasispecies. The population diversity first suffers striking expansions in the presence of mutagens and then compressions either when the presence of the mutagenic analogue was discontinued or when a mutation that decreased sensitivity to a mutagen was selected. The pattern of mutations found in the populations was in agreement with the behavior of the corresponding nucleotide analogues with FMDV in vitro. Mutations accumulated at preferred genomic sites, and dn/ds ratios indicate the operation of negative (or purifying) selection in populations subjected to mutagenesis. No evidence of unusually elevated genetic distances has been obtained for FMDV populations approaching extinction. Phylogenetic and PAQ analysis provide adequate procedures to describe the evolution of viral sequences subjected to lethal mutagenesis. These methods define the changes of intra-population structure more precisely than mutation frequencies and Shannon entropies. PAQ is very sensitive to variations of intrapopulation genetic distances. Strong negative (or purifying) selection operates in FMDV populations subjected to enhanced mutagenesis. The quantifications provide evidence that extinction does not imply unusual increases of intrapopulation complexity, in support of the lethal defection model of virus extinction.
Selective Expansion of Memory CD4+ T cells By Mitogenic Human CD28 Generates Inflammatory Cytokines and Regulatory T cells

PubMed Central

Singh, Manisha; Basu, Sreemanti; Camell, Christina; Couturier, Jacob; Nudelman, Rodolfo J.; Medina, Miguel A.; Rodgers, John R.; Lewis, Dorothy E.

2009-01-01

Co-stimulatory signals are important for development of effector and regulatory T cells. In this case, CD28 signaling is usually considered inert in the absence of signaling through the TCR. By contrast, mitogenic rat CD28 mAbs reportedly expand regulatory T cells without TCR stimulation. We found that a commercially available human CD28 mAb (ANC28) stimulated PBMCs without TCR co-ligation or cross-linking; ANC28 selectively expanded CD4+CD25+FoxP3−(T effector) and CD4+CD25+FoxP3+ (Treg) cells. ANC28 stimulated the CD45RO+ CD4+ (memory) population whereas CD45RA+CD4+ (naïve) cells did not respond. ANC28 also induced inflammatory cytokines. Treg induced by ANC28 retain the Treg phenotype longer than did co-stimulated Treg. Treg induced by ANC28 suppressed CD25− T cells through a contact-dependent mechanism. Purity influenced the response of CD4+CD25+ cells because bead-purified CD4+CD25+ cells (85–90% pure) responded strongly to ANC28, whereas 98% pure FACS-sorted CD4+CD25 bright (T-reg) did not respond. Purified CD4+CD25int cells responded similarly to the bead-purified CD4+CD25+ cells. Thus, pre-activated CD4+ T cells (CD25int) respond to ANC28 rather than Treg (CD25bright). The ability of ANC28 to expand both effectors producing inflammatory cytokines as well as suppressive regulatory T cells might be useful for ex vivo expansion of therapeutic T cells. PMID:18446791
Molecular Evolution of the Neural Crest Regulatory Network in Ray-Finned Fish

PubMed Central

Kratochwil, Claudius F.; Geissler, Laura; Irisarri, Iker; Meyer, Axel

2015-01-01

Abstract Gene regulatory networks (GRN) are central to developmental processes. They are composed of transcription factors and signaling molecules orchestrating gene expression modules that tightly regulate the development of organisms. The neural crest (NC) is a multipotent cell population that is considered a key innovation of vertebrates. Its derivatives contribute to shaping the astounding morphological diversity of jaws, teeth, head skeleton, or pigmentation. Here, we study the molecular evolution of the NC GRN by analyzing patterns of molecular divergence for a total of 36 genes in 16 species of bony fishes. Analyses of nonsynonymous to synonymous substitution rate ratios (dN/dS) support patterns of variable selective pressures among genes deployed at different stages of NC development, consistent with the developmental hourglass model. Model-based clustering techniques of sequence features support the notion of extreme conservation of NC-genes across the entire network. Our data show that most genes are under strong purifying selection that is maintained throughout ray-finned fish evolution. Late NC development genes reveal a pattern of increased constraints in more recent lineages. Additionally, seven of the NC-genes showed signs of relaxation of purifying selection in the famously species-rich lineage of cichlid fishes. This suggests that NC genes might have played a role in the adaptive radiation of cichlids by granting flexibility in the development of NC-derived traits—suggesting an important role for NC network architecture during the diversification in vertebrates. PMID:26475317
Production and Evaluation of a Purified Adenovirus Group-Specific (Hexon) Antigen for Use in the Diagnostic Complement Fixation Test

PubMed Central

Dowdle, W. R.; Lambriex, M.; Hierholzer, J. C.

1971-01-01

A simple procedure for the production of large volumes of purified adenovirus group-specific complement-fixing (CF) (hexon) antigen by selective adsorption to and elution from CaHPO4 is described. Results of immunodiffusion tests, electrophoresis, electron microscopy, and tests for hemagglutination and infectivity indicate that the purified antigen consisted of a single virus component (hexon). The purified product contained little host materials. Unlike the crude virus harvest usually employed for serodiagnostic CF tests, the purified antigen demonstrated no anticomplementary activity and did not develop such activity during storage. The purified antigen was equal to or slightly more sensitive than crude virus harvests for serodiagnosis of adenovirus infections. Images PMID:4325021
Enterocin P Selectively Dissipates the Membrane Potential of Enterococcus faecium T136

PubMed Central

Herranz, C.; Chen, Y.; Chung, H.-J.; Cintas, L. M.; Hernández, P. E.; Montville, T. J.; Chikindas, M. L.

2001-01-01

Enterocin P is a pediocin-like, broad-spectrum bacteriocin which displays a strong inhibitory activity against Listeria monocytogenes. The bacteriocin was purified from the culture supernatant of Enterococcus faecium P13, and its molecular mechanism of action against the sensitive strain E. faecium T136 was evaluated. Although enterocin P caused significant reduction of the membrane potential (ΔΨ) and the intracellular ATP pool of the indicator organism, the pH gradient (ΔpH) component of the proton motive force (Δp) was not dissipated. By contrast, enterocin P caused carboxyfluorescein efflux from E. faecium T136-derived liposomes. PMID:11282622
Expression level, cellular compartment and metabolic network position all influence the average selective constraint on mammalian enzymes

PubMed Central

2011-01-01

Background A gene's position in regulatory, protein interaction or metabolic networks can be predictive of the strength of purifying selection acting on it, but these relationships are neither universal nor invariably strong. Following work in bacteria, fungi and invertebrate animals, we explore the relationship between selective constraint and metabolic function in mammals. Results We measure the association between selective constraint, estimated by the ratio of nonsynonymous (Ka) to synonymous (Ks) substitutions, and several, primarily metabolic, measures of gene function. We find significant differences between the selective constraints acting on enzyme-coding genes from different cellular compartments, with the nucleus showing higher constraint than genes from either the cytoplasm or the mitochondria. Among metabolic genes, the centrality of an enzyme in the metabolic network is significantly correlated with Ka/Ks. In contrast to yeasts, gene expression magnitude does not appear to be the primary predictor of selective constraint in these organisms. Conclusions Our results imply that the relationship between selective constraint and enzyme centrality is complex: the strength of selective constraint acting on mammalian genes is quite variable and does not appear to exclusively follow patterns seen in other organisms. PMID:21470417
On The Relationship between Suspended Solids of Different Size, the Observed Boundary Flux and Rejection Values for Membranes Treating a Civil Wastewater Stream.

PubMed

Stoller, Marco; Pulido, Javier Miguel Ochando; Palma, Luca Di

2014-08-04

Membrane fouling is one of the main issues in membrane processes, leading to a progressive decrease of permeability. High fouling rates strongly reduce the productivity of the membrane plant, and negatively affect the surviving rate of the membrane modules, especially when real wastewater is treated. On the other hand, since selectivity must meet certain target requirements, fouling may lead to unexpected selectivity improvements due to the formation of an additional superficial layer formed of foulants and that act like a selective secondary membrane layer. In this case, a certain amount of fouling may be profitable to the point where selectivity targets were reached and productivity is not significantly affected. In this work, the secondary clarifier of a step sludge recirculation bioreactor treating municipal wastewater was replaced by a membrane unit, aiming at recovering return sludge and producing purified water. Fouling issues of such a system were checked by boundary flux measurements. A simple model for the description of the observed productivity and selectivity values as a function of membrane fouling is proposed.
On The Relationship between Suspended Solids of Different Size, the Observed Boundary Flux and Rejection Values for Membranes Treating a Civil Wastewater Stream

PubMed Central

Stoller, Marco; Pulido, Javier Miguel Ochando; Di Palma, Luca

2014-01-01

Membrane fouling is one of the main issues in membrane processes, leading to a progressive decrease of permeability. High fouling rates strongly reduce the productivity of the membrane plant, and negatively affect the surviving rate of the membrane modules, especially when real wastewater is treated. On the other hand, since selectivity must meet certain target requirements, fouling may lead to unexpected selectivity improvements due to the formation of an additional superficial layer formed of foulants and that act like a selective secondary membrane layer. In this case, a certain amount of fouling may be profitable to the point where selectivity targets were reached and productivity is not significantly affected. In this work, the secondary clarifier of a step sludge recirculation bioreactor treating municipal wastewater was replaced by a membrane unit, aiming at recovering return sludge and producing purified water. Fouling issues of such a system were checked by boundary flux measurements. A simple model for the description of the observed productivity and selectivity values as a function of membrane fouling is proposed. PMID:25093867
[Screening and antibacterial function of Bacillus amyloliquefaciens X030].

PubMed

He, Hao; Zhu, Yingling; Chi, Liqing; Zhao, Zizhao; Wang, Ting; Zuo, Mingxing; Zhang, Tong; Zhou, Fengjuan; Xia, Liqiu; Ding, Xuezhi

2015-09-04

We isolated 339 bacillus strains from 72 soil samples all over the country, then purified their antimicrobial compounds and studied the antibacterial activity, to enrich bacillus resources and explore their second metabolites. A bacillus strain with strong antibacterial activity was selected by dilution plate and water bath heating from a soil sample from a peanut plantation in Henan Province; this strain was identified according to morphological observation, physiological and biochemical characteristics, and consequences of 16S rRNA homologous analysis. Antibacterial compound from the identified strain, Bacillus amyloliquefaciens X030, was separated and purified by acetone precipitation, Sephadex chromatography, C18 reverse phase column chromatography. Its molecular weight was analyzed by LC-MS/MS. The antibacterial activity was characterized by disc diffusion and plate two-way cultivation. Bacillus amyloliquefaciens was isolated that not only has antibacterial activity against Staphylococcus aureus, Candida albican and Saccharomycetes; but also against Pyriculariaoryzae, Chili pointed cell anthrax, Gloeosporium eriobotryae speg and Phytophthora parasitica. The compound was confirmed as polypeptide. Bacillus amyloliquefaciens X030 can produce a polypeptide that inhibits pathogenic bacteria and plant pathogenic fungi.
Comparative transcriptome resources of two Dysosma species (Berberidaceae) and molecular evolution of the CYP719A gene in Podophylloideae.

PubMed

Mao, Yunrui; Zhang, Yonghua; Xu, Chuan; Qiu, Yingxiong

2016-01-01

Dysosma species (Berberidaceae, Podophylloideae) are of great medicinal pharmacogenetic importance and used as model systems to study the drivers and mechanisms of species diversification of temperate plants in East Asia. Recently, we have sequenced the transcriptome of the low-elevation D. versipellis. In this study, we sequenced the transcriptome of the high-elevation D. aurantiocaulis and used comparative genomic approaches to investigate the transcriptome evolution of the two species. We retrieved 53,929 unigenes from D. aurantiocaulis by de novo transcriptome assemblies using the Illumina HiSeq 2000 platform. Comparing the transcriptomes of both species, we identified 4593 orthologs. Estimation of Ka/Ks ratios for 3126 orthologs revealed that none had a Ka/Ks significantly greater than 1, whereas 1273 (Ka/Ks < 0.5, P < 0.05) were inferred to be under purifying selection. A total of 51 primer pairs were successfully designed from 461 EST-SSRs contained in 4593 orthologs. Marker validation assay revealed that 26 (51%) and 41 (80.4%) produced clear fragments with the expected sizes in all Podophylloideae species. Specifically, 19 different sequences of CYP719A were identified from PCR-amplified genomic DNA of all 12 species of Podophylloideae using primers designed from the assembled transcripts. The data further indicated that CYP719A was likely subject to strong selective constraints maintaining only one copy per genome. In Dysosma, there was relaxed purifying selection or more positive selection for high-elevation species. Overall, this study has generated a wealth of molecular resources potentially useful for pharmacogenetic and evolutionary studies in Dysosma and allied taxa. © 2015 John Wiley & Sons Ltd.
Topology of evolving, mutagenized viral populations: quasispecies expansion, compression, and operation of negative selection

PubMed Central

2008-01-01

Background The molecular events and evolutionary forces underlying lethal mutagenesis of virus (or virus extinction through an excess of mutations) are not well understood. Here we apply for the first time phylogenetic methods and Partition Analysis of Quasispecies (PAQ) to monitor genetic distances and intra-population structures of mutant spectra of foot-and-mouth disease virus (FMDV) quasispecies subjected to mutagenesis by base and nucleoside analogues. Results Phylogenetic and PAQ analyses have revealed a highly dynamic variation of intrapopulation diversity of FMDV quasispecies. The population diversity first suffers striking expansions in the presence of mutagens and then compressions either when the presence of the mutagenic analogue was discontinued or when a mutation that decreased sensitivity to a mutagen was selected. The pattern of mutations found in the populations was in agreement with the behavior of the corresponding nucleotide analogues with FMDV in vitro. Mutations accumulated at preferred genomic sites, and dn/ds ratios indicate the operation of negative (or purifying) selection in populations subjected to mutagenesis. No evidence of unusually elevated genetic distances has been obtained for FMDV populations approaching extinction. Conclusion Phylogenetic and PAQ analysis provide adequate procedures to describe the evolution of viral sequences subjected to lethal mutagenesis. These methods define the changes of intra-population structure more precisely than mutation frequencies and Shannon entropies. PAQ is very sensitive to variations of intrapopulation genetic distances. Strong negative (or purifying) selection operates in FMDV populations subjected to enhanced mutagenesis. The quantifications provide evidence that extinction does not imply unusual increases of intrapopulation complexity, in support of the lethal defection model of virus extinction. PMID:18637173
Modular Organization of Residue-Level Contacts Shapes the Selection Pressure on Individual Amino Acid Sites of Ribosomal Proteins.

PubMed

Mallik, Saurav; Kundu, Sudip

2017-04-01

Understanding the molecular evolution of macromolecular complexes in the light of their structure, assembly, and stability is of central importance. Here, we address how the modular organization of native molecular contacts shapes the selection pressure on individual residue sites of ribosomal complexes. The bacterial ribosomal complex is represented as a residue contact network where nodes represent amino acid/nucleotide residues and edges represent their van der Waals interactions. We find statistically overrepresented native amino acid-nucleotide contacts (OaantC, one amino acid contacts one or multiple nucleotides, internucleotide contacts are disregarded). Contact number is defined as the number of nucleotides contacted. Involvement of individual amino acids in OaantCs with smaller contact numbers is more random, whereas only a few amino acids significantly contribute to OaantCs with higher contact numbers. An investigation of structure, stability, and assembly of bacterial ribosome depicts the involvement of these OaantCs in diverse biophysical interactions stabilizing the complex, including high-affinity protein-RNA contacts, interprotein cooperativity, intersubunit bridge, packing of multiple ribosomal RNA domains, etc. Amino acid-nucleotide constituents of OaantCs with higher contact numbers are generally associated with significantly slower substitution rates compared with that of OaantCs with smaller contact numbers. This evolutionary rate heterogeneity emerges from the strong purifying selection pressure that conserves the respective amino acid physicochemical properties relevant to the stabilizing interaction with OaantC nucleotides. An analysis of relative molecular orientations of OaantC residues and their interaction energetics provides the biophysical ground of purifying selection conserving OaantC amino acid physicochemical properties. © The Author(s) 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.
Recombinant production of enzymatically active male contraceptive drug target hTSSK2 - Localization of the TSKS domain phosphorylated by TSSK2.

PubMed

Shetty, Jagathpala; Sinville, Rondedrick; Shumilin, Igor A; Minor, Wladek; Zhang, Jianhai; Hawkinson, Jon E; Georg, Gunda I; Flickinger, Charles J; Herr, John C

2016-05-01

The testis-specific serine/threonine kinase 2 (TSSK2) has been proposed as a candidate male contraceptive target. Development of a selective inhibitor for this kinase first necessitates the production of highly purified, soluble human TSSK2 and its substrate, TSKS, with high yields and retention of biological activity for crystallography and compound screening. Strategies to produce full-length, soluble, biologically active hTSSK2 in baculovirus expression systems were tested and refined. Soluble preparations of TSSK2 were purified by immobilized-metal affinity chromatography (IMAC) followed by gel filtration chromatography. The biological activities of rec.hTSSK2 were verified by in vitro kinase and mobility shift assays using bacterially produced hTSKS (isoform 2), casein, glycogen synthase peptide (GS peptide) and various TSKS peptides as target substrates. Purified recombinant hTSSK2 showed robust kinase activity in the in vitro kinase assay by phosphorylating hTSKS isoform 2 and casein. The ATP Km values were similar for highly and partially purified fractions of hTSSK2 (2.2 and 2.7 μM, respectively). The broad spectrum kinase inhibitor staurosporine was a potent inhibitor of rec.hTSSK2 (IC50 = 20 nM). In vitro phosphorylation experiments carried out with TSKS (isoform 1) fragments revealed particularly strong phosphorylation of a recombinant N-terminal region representing aa 1-150 of TSKS, indicating that the N-terminus of human TSKS is phosphorylated by human TSSK2. Production of full-length enzymatically active recombinant TSSK2 kinase represents the achievement of a key benchmark for future discovery of TSSK inhibitors as male contraceptive agents. Copyright © 2016 Elsevier Inc. All rights reserved.
Evolution and functional divergence of the anoctamin family of membrane proteins

PubMed Central

2010-01-01

Background The anoctamin family of transmembrane proteins are found in all eukaryotes and consists of 10 members in vertebrates. Ano1 and ano2 were observed to have Ca2+ activated Cl- channel activity. Recent findings however have revealed that ano6, and ano7 can also produce chloride currents, although with different properties. In contrast, ano9 and ano10 suppress baseline Cl- conductance when co-expressed with ano1 thus suggesting that different anoctamins can interfere with each other. In order to elucidate intrinsic functional diversity, and underlying evolutionary mechanism among anoctamins, we performed comprehensive bioinformatics analysis of anoctamin gene family. Results Our results show that anoctamin protein paralogs evolved from several gene duplication events followed by functional divergence of vertebrate anoctamins. Most of the amino acid replacements responsible for the functional divergence were fixed by adaptive evolution and this seem to be a common pattern in anoctamin gene family evolution. Strong purifying selection and the loss of many gene duplication products indicate rigid structure-function relationships among anoctamins. Conclusions Our study suggests that anoctamins have evolved by series of duplication events, and that they are constrained by purifying selection. In addition we identified a number of protein domains, and amino acid residues which contribute to predicted functional divergence. Hopefully, this work will facilitate future functional characterization of the anoctamin membrane protein family. PMID:20964844
Molecular evolution of the odorant and gustatory receptor genes in lepidopteran insects: implications for their adaptation and speciation.

PubMed

Engsontia, Patamarerk; Sangket, Unitsa; Chotigeat, Wilaiwan; Satasook, Chutamas

2014-08-01

Lepidoptera (comprised of butterflies and moths) is one of the largest groups of insects, including more than 160,000 described species. Chemoreception plays important roles in the adaptation of these species to a wide range of niches, e.g., plant hosts, egg-laying sites, and mates. This study investigated the molecular evolution of the lepidopteran odorant (Or) and gustatory receptor (Gr) genes using recently identified genes from Bombyx mori, Danaus plexippus, Heliconius melpomene, Plutella xylostella, Heliothis virescens, Manduca sexta, Cydia pomonella, and Spodoptera littoralis. A limited number of cases of large lineage-specific gene expansion are observed (except in the P. xylostella lineage), possibly due to selection against tandem gene duplication. There has been strong purifying selection during the evolution of both lepidopteran odorant and gustatory genes, as shown by the low ω values estimated through CodeML analysis, ranging from 0.0093 to 0.3926. However, purifying selection has been relaxed on some amino acid sites in these receptors, leading to sequence divergence, which is a precursor of positive selection on these sequences. Signatures of positive selection were detected only in a few loci from the lineage-specific analysis. Estimation of gene gains and losses suggests that the common ancestor of the Lepidoptera had fewer Or genes compared to extant species and an even more reduced number of Gr genes, particularly within the bitter receptor clade. Multiple gene gains and a few gene losses occurred during the evolution of Lepidoptera. Gene family expansion may be associated with the adaptation of lepidopteran species to plant hosts, especially after angiosperm radiation. Phylogenetic analysis of the moth sex pheromone receptor genes suggested that chromosomal translocations have occurred several times. New sex pheromone receptors have arisen through tandem gene duplication. Positive selection was detected at some amino acid sites predicted to be in the extracellular and transmembrane regions of the newly duplicated genes, which might be associated with the evolution of the new pheromone receptors.
Purifying arsenic and fluoride-contaminated water by a novel graphene-based nanocomposite membrane of enhanced selectivity and sustained flux.

PubMed

Pal, Madhubonti; Mondal, Mrinal Kanti; Paine, Tapan Kanti; Pal, Parimal

2018-06-01

A novel graphene-based nanocomposite membrane was synthesized by interfacial polymerization (IP) through chemical bonding of the graphene oxide (GO) layer to polyethersulfone surface. Detailed characterization of the composite membrane through AFM, SEM, ATR-FTIR, XRD analysis, and Raman spectroscopy indicates strong potential of the membrane in highly selective removal of the toxic contaminants like arsenic and fluoride while permeating the essential minerals like calcium and magnesium. This makes the membrane suitable for production of safe drinking water from contaminated water. The membrane applied in a flat-sheet cross-flow module succeeded in removal of more than 98% arsenic and around 80% fluoride from contaminated water while selectively retaining the useful calcium and magnesium minerals in drinking water. A sustained pure water flux of around 150 LMH (liter per square meter per hour) during operation over long hours (> 150 h) with only 3-5% drop in flux indicates antifouling character of the membrane module.
Purifying Selection and Birth-and-Death Evolution in the Class II Hydrophobin Gene Families of the Ascomycete Trichoderma/Hypocrea

DOE Office of Scientific and Technical Information (OSTI.GOV)

kubicek, Christian P.; Baker, Scott E.; Gamauf, Christian

2008-01-10

Hydrophobins are proteins containing eight conserved cysteine residues that occur uniquely in mycelial fungi, where their main function is to confer hydrophobicity to fungal surfaces in contact with air and during attachment of hyphae to hydrophobic surfaces of hosts, symbiotic partners or of themselves resulting in morphogenetic signals. Based on their hydropathy patterns and their solubility characteristics, they are classified in class I and class II hydrophobins, the latter being found only in ascomycetes. Here we have investigated the mechanisms driving the evolution of the class II hydrophobins in nine species of the mycoparasitic ascomycetous genus Trichoderma/Hypocrea, using three fullymore » sequenced genomes (H. jecorina=T. reesei, H. atroviridis=T. atroviride; H. virens=T. virens) and a total of 14.000 ESTs of six others (T. asperellum, H. lixii=T. harzianum, T. aggressivum var. europeae, T. longibrachiatum, T. cf. viride). The former three contained six, ten and nine members, which is the highest number found in any other ascomycete so far. They all showed the conserved four beta-strands/one helix structure, which is stabilized by four disulfide bonds. In addition, a small number of these HFBs contained an extended N-terminus rich in either praline and aspartate, or glycine-asparagine. Phylogenetic analysis reveals a mosaic of terminal clades contain duplicated genes and shows only three reasonably supported clades. Calculation of the ratio of differences in synonymous vs. non-synonymous nucleotide substitutions provides evidence for strong purifying selection (KS/Ka >> 1). A genome database search for class II HFBs from other ascomycetes retrieved a much smaller number of hydrophobins (2-4) from each species, and most of them were from Pyrenomycetes. A combined phylogeny of these sequences with those of Trichoderma showed that the Trichoderma HFBs mostly formed their own clades, whereas those of other pyrenomycetes occured in shared clades. Our study shows that the genus Trichoderma/Hypocrea has a proliferated arsenal of class II hydrophobins which arose by purifying selection and birth-and-death evolution.« less
Characterization of the transcriptome, nucleotide sequence polymorphism, and natural selection in the desert adapted mouse Peromyscus eremicus.

PubMed

MacManes, Matthew D; Eisen, Michael B

2014-01-01

As a direct result of intense heat and aridity, deserts are thought to be among the most harsh of environments, particularly for their mammalian inhabitants. Given that osmoregulation can be challenging for these animals, with failure resulting in death, strong selection should be observed on genes related to the maintenance of water and solute balance. One such animal, Peromyscus eremicus, is native to the desert regions of the southwest United States and may live its entire life without oral fluid intake. As a first step toward understanding the genetics that underlie this phenotype, we present a characterization of the P. eremicus transcriptome. We assay four tissues (kidney, liver, brain, testes) from a single individual and supplement this with population level renal transcriptome sequencing from 15 additional animals. We identified a set of transcripts undergoing both purifying and balancing selection based on estimates of Tajima's D. In addition, we used the branch-site test to identify a transcript-Slc2a9, likely related to desert osmoregulation-undergoing enhanced selection in P. eremicus relative to a set of related non-desert rodents.
A Conserved Cytochrome P450 Evolved in Seed Plants Regulates Flower Maturation.

PubMed

Liu, Zhenhua; Boachon, Benoît; Lugan, Raphaël; Tavares, Raquel; Erhardt, Mathieu; Mutterer, Jérôme; Demais, Valérie; Pateyron, Stéphanie; Brunaud, Véronique; Ohnishi, Toshiyuki; Pencik, Ales; Achard, Patrick; Gong, Fan; Hedden, Peter; Werck-Reichhart, Danièle; Renault, Hugues

2015-12-07

Global inspection of plant genomes identifies genes maintained in low copies across taxa and under strong purifying selection, which are likely to have essential functions. Based on this rationale, we investigated the function of the low-duplicated CYP715 cytochrome P450 gene family that appeared early in seed plants and evolved under strong negative selection. Arabidopsis CYP715A1 showed a restricted tissue-specific expression in the tapetum of flower buds and in the anther filaments upon anthesis. cyp715a1 insertion lines showed a strong defect in petal development, and transient alteration of pollen intine deposition. Comparative expression analysis revealed the downregulated expression of genes involved in pollen development, cell wall biogenesis, hormone homeostasis, and floral sesquiterpene biosynthesis, especially TPS21 and several key genes regulating floral development such as MYB21, MYB24, and MYC2. Accordingly, floral sesquiterpene emission was suppressed in the cyp715a1 mutants. Flower hormone profiling, in addition, indicated a modification of gibberellin homeostasis and a strong disturbance of the turnover of jasmonic acid derivatives. Petal growth was partially restored by the active gibberellin GA3 or the functional analog of jasmonoyl-isoleucine, coronatine. CYP715 appears to function as a key regulator of flower maturation, synchronizing petal expansion and volatile emission. It is thus expected to be an important determinant of flower-insect interaction. Copyright © 2015 The Author. Published by Elsevier Inc. All rights reserved.
Recombinant Mip-PilE-FlaA dominant epitopes vaccine candidate against Legionella pneumophila.

PubMed

He, Jinlei; Huang, Fan; Chen, Han; Chen, Qiwei; Zhang, Junrong; Li, Jiao; Chen, Dali; Chen, Jianping

2017-06-01

Legionella pneumophila is the main causative agent of Legionnaires' disease, which is a severe multi-system disease with pneumonia as the primary manifestation. We designed a recombinant Mip-PilE-FlaA dominant epitopes vaccine against Legionella pneumophila to prevent the disease and evaluated its immunogenicity and protective immunity. The protein structures of Mip, PilE and FlaA were analyzed using a computer, and the gene sequences of the dominant epitopes of the three proteins were selected to construct and optimize the vaccine. The optimized mip, pilE, flaA and recombinant mip-pilE-flaA gene sequences were cloned, expressed and purified. The purified proteins were used as dominant epitopes vaccines to immunize BALB/c mice and determine the protective immunity and immunogenicity of these purified proteins. The identification confirmed that the recombinant mip-pilE-flaA was successfully cloned and expressed. ELISA revealed that the Mip-PilE-FlaA group produced the highest IgG response, and this protein may considerably improve the production of some cytokines in BALB/c mice. Histopathology analyses of lungs from mice immunized with Mip-PilE-FlaA revealed a certain protective effect. Our work demonstrated that the recombinant dominant epitopes of Mip-PilE-FlaA exhibited strong immunogenicity and immune protection, and this protein may be an efficient epitopes vaccine candidate against Legionella pneumophila. Copyright © 2017 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.

Contemporary evolution of resistance at the major insecticide target site gene Ace-1 by mutation and copy number variation in the malaria mosquito Anopheles gambiae

PubMed Central

Weetman, David; Mitchell, Sara N; Wilding, Craig S; Birks, Daniel P; Yawson, Alexander E; Essandoh, John; Mawejje, Henry D; Djogbenou, Luc S; Steen, Keith; Rippon, Emily J; Clarkson, Christopher S; Field, Stuart G; Rigden, Daniel J; Donnelly, Martin J

2015-01-01

Functionally constrained genes are ideal insecticide targets because disruption is often fatal, and resistance mutations are typically costly. Synaptic acetylcholinesterase (AChE) is an essential neurotransmission enzyme targeted by insecticides used increasingly in malaria control. In Anopheles and Culex mosquitoes, a glycine–serine substitution at codon 119 of the Ace-1 gene confers both resistance and fitness costs, especially for 119S/S homozygotes. G119S in Anopheles gambiae from Accra (Ghana) is strongly associated with resistance, and, despite expectations of cost, resistant 119S alleles are increasing significantly in frequency. Sequencing of Accra females detected only a single Ace-1 119S haplotype, whereas 119G diversity was high overall but very low at non-synonymous sites, evidence of strong purifying selection driven by functional constraint. Flanking microsatellites showed reduced diversity, elevated linkage disequilibrium and high differentiation of 119S, relative to 119G homozygotes across up to two megabases of the genome. Yet these signals of selection were inconsistent and sometimes weak tens of kilobases from Ace-1. This unexpected finding is attributable to apparently ubiquitous amplification of 119S alleles as part of a large copy number variant (CNV) far exceeding the size of the Ace-1 gene, whereas 119G alleles were unduplicated. Ace-1 CNV was detectable in archived samples collected when the 119S allele was rare in Ghana. Multicopy amplification of resistant alleles has not been observed previously and is likely to underpin the recent increase in 119S frequency. The large CNV compromised localization of the strong selective sweep around Ace-1, emphasizing the need to integrate CNV analysis into genome scans for selection. PMID:25865270
Anti-inflammatory, cyclooxygenase (COX)-2, COX-1 inhibitory, and free radical scavenging effects of Rumex nepalensis.

PubMed

Gautam, Raju; Karkhile, Kailas V; Bhutani, Kamlesh K; Jachak, Sanjay M

2010-10-01

Evaluation of the topical anti-inflammatory activity of chloroform and ethyl acetate extracts of RUMEX NEPALENSIS roots in a TPA-induced acute inflammation mouse model demonstrated a significant reduction in ear edema. The extracts were further tested on purified enzymes for COX-1 and COX-2 inhibition to elucidate their mechanism of action, and a strong inhibition was observed. Six anthraquinones and two naphthalene derivatives were isolated from the ethyl acetate extract. Among the isolated compounds, emodin was found to be a potent inhibitor with slight selectivity towards COX-2, and nepodin exhibited selectivity towards COX-1. Emodin, endocrocin, and nepodin also exhibited significant topical anti-inflammatory activity in mice. Interestingly, nepodin showed better radical scavenging activity than trolox and ascorbic acid against DPPH and ABTS radicals. The strong radical scavenging activity of chloroform and ethyl acetate extracts could be explained by the presence of nepodin as well as by the high phenolic content of the ethyl acetate extract. Thus, the anti-inflammatory effect of R. NEPALENSIS roots was assumed to be mediated through COX inhibition by anthraquinones and naphthalene derivatives and through the radical scavenging activities of naphthalene derivatives. © Georg Thieme Verlag KG Stuttgart · New York.
Genetic diversity of the HLA-G coding region in Amerindian populations from the Brazilian Amazon: a possible role of natural selection.

PubMed

Mendes-Junior, C T; Castelli, E C; Meyer, D; Simões, A L; Donadi, E A

2013-12-01

HLA-G has an important role in the modulation of the maternal immune system during pregnancy, and evidence that balancing selection acts in the promoter and 3'UTR regions has been previously reported. To determine whether selection acts on the HLA-G coding region in the Amazon Rainforest, exons 2, 3 and 4 were analyzed in a sample of 142 Amerindians from nine villages of five isolated tribes that inhabit the Central Amazon. Six previously described single-nucleotide polymorphisms (SNPs) were identified and the Expectation-Maximization (EM) and PHASE algorithms were used to computationally reconstruct SNP haplotypes (HLA-G alleles). A new HLA-G allele, which originated in Amerindian populations by a crossing-over event between two widespread HLA-G alleles, was identified in 18 individuals. Neutrality tests evidenced that natural selection has a complex part in the HLA-G coding region. Although balancing selection is the type of selection that shapes variability at a local level (Native American populations), we have also shown that purifying selection may occur on a worldwide scale. Moreover, the balancing selection does not seem to act on the coding region as strongly as it acts on the flanking regulatory regions, and such coding signature may actually reflect a hitchhiking effect.
Natural selection reduced diversity on human y chromosomes.

PubMed

Wilson Sayres, Melissa A; Lohmueller, Kirk E; Nielsen, Rasmus

2014-01-01

The human Y chromosome exhibits surprisingly low levels of genetic diversity. This could result from neutral processes if the effective population size of males is reduced relative to females due to a higher variance in the number of offspring from males than from females. Alternatively, selection acting on new mutations, and affecting linked neutral sites, could reduce variability on the Y chromosome. Here, using genome-wide analyses of X, Y, autosomal and mitochondrial DNA, in combination with extensive population genetic simulations, we show that low observed Y chromosome variability is not consistent with a purely neutral model. Instead, we show that models of purifying selection are consistent with observed Y diversity. Further, the number of sites estimated to be under purifying selection greatly exceeds the number of Y-linked coding sites, suggesting the importance of the highly repetitive ampliconic regions. While we show that purifying selection removing deleterious mutations can explain the low diversity on the Y chromosome, we cannot exclude the possibility that positive selection acting on beneficial mutations could have also reduced diversity in linked neutral regions, and may have contributed to lowering human Y chromosome diversity. Because the functional significance of the ampliconic regions is poorly understood, our findings should motivate future research in this area.
Natural Selection Reduced Diversity on Human Y Chromosomes

PubMed Central

Wilson Sayres, Melissa A.; Lohmueller, Kirk E.; Nielsen, Rasmus

2014-01-01

The human Y chromosome exhibits surprisingly low levels of genetic diversity. This could result from neutral processes if the effective population size of males is reduced relative to females due to a higher variance in the number of offspring from males than from females. Alternatively, selection acting on new mutations, and affecting linked neutral sites, could reduce variability on the Y chromosome. Here, using genome-wide analyses of X, Y, autosomal and mitochondrial DNA, in combination with extensive population genetic simulations, we show that low observed Y chromosome variability is not consistent with a purely neutral model. Instead, we show that models of purifying selection are consistent with observed Y diversity. Further, the number of sites estimated to be under purifying selection greatly exceeds the number of Y-linked coding sites, suggesting the importance of the highly repetitive ampliconic regions. While we show that purifying selection removing deleterious mutations can explain the low diversity on the Y chromosome, we cannot exclude the possibility that positive selection acting on beneficial mutations could have also reduced diversity in linked neutral regions, and may have contributed to lowering human Y chromosome diversity. Because the functional significance of the ampliconic regions is poorly understood, our findings should motivate future research in this area. PMID:24415951
Adsorption of lead onto smectite from aqueous solution.

PubMed

Mhamdi, M; Galai, H; Mnasri, N; Elaloui, E; Trabelsi-Ayadi, M

2013-03-01

The purpose of this research is to study the effect of a new method of adsorption using membrane filtration to determine the maximum amount of lead adsorbed by clay and investigate the behavior of the clay after adsorption of the said metal. Treatment of wastewater contaminated with heavy metals depends on the characteristics of the effluent, the amount of final discharge, the cost of treatment, and the compatibility of the treatment process. The process of adsorption of heavy metals by clays may be a simple, selective, and economically viable alternative to the conventional physical-chemical treatment. This is justified by the importance of the surface developed by this material, the presence of negative charges on the said surface, the possibility of ion exchange taking place, and its wide availability in nature. The removal of lead from wastewater was studied by using the adsorption technique and using clay as the adsorbent. A method was optimized for adsorption through a membrane approaching natural adsorption. This new method is simple, selective, and the lead adsorption time is about 3 days. The various properties of clay were determined. It was observed that the cation exchange capacity of the clay was 56 meq/100 g of hydrated clay for the raw sample and 82 meq/100 g for the purified sample. The total surface area determined by the methylene blue method was equal to 556 and 783 m(2)/g for the raw and purified samples, respectively. The adsorption kinetics depends on several parameters. The Pb(II) clay, obeys the Langmuir, Freundlich, and the Elovich adsorption isotherms with high regression coefficients. The use of this adsorbent notably decreases the cost of treatment. It was concluded that clay shows a strong adsorption capacity on Pb(II), the maximum interaction occurring with purified clay treated at high concentration of lead. It is proposed that this adsorption through a membrane be extended for the treatment of effluents containing other metals.
High-yields heterologous production of the novel Aspergillus fumigatus elastase inhibitor AFUEI in Aspergillus oryzae.

PubMed

Yamashita, Nobuo; Komori, Yumiko; Okumura, Yoshiyuki; Uchiya, Kei-Ichi; Matsui, Takeshi; Nishimura, Akira; Ogawa, Kenji; Nikai, Toshiaki

2011-08-01

AFUEI, an elastase inhibitor produced by Aspergillus fumigatus strongly inhibits the elastolytic activity of A. fumigatus etc. To purify AFUEI, we constructed a strain that overproduces AFUEI by introducing the gene encoding AFUEI (Genbank accession no. AB546725) under control of the amyB promoter into the heterologous host Aspergillus oryzae. A. oryzae TF-4 displayed strong elastase inhibitory activity and produced considerably more AFUEI than that of A. fumigatus. Furthermore, AFUEI could be purified using culture broth and single ultrafiltration (UF) treatment, allowing for the effective production of AFUEI for use in clinical trials. Copyright © 2011 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.
Induction of Pro-Angiogenic Factors by Pregnancy-Specific Glycoproteins and Studies on Receptor Usage

DTIC Science & Technology

2008-01-01

expression vector. The purified bacoluvirus DNA, containing PSG11, was purified and used to transfect Spodoptera frugiperda (Sf-9) cells (Invitrogen) to...proteins in Spodoptera frugiperda cells using biotin acceptor peptides. Anal Biochem, 1998. 262(2): p. 122-8. 175. Hirt, B., Selective extraction of
Implications of sex-specific selection for the genetic basis of disease.

PubMed

Morrow, Edward H; Connallon, Tim

2013-12-01

Mutation and selection are thought to shape the underlying genetic basis of many common human diseases. However, both processes depend on the context in which they occur, such as environment, genetic background, or sex. Sex has widely known effects on phenotypic expression of genotype, but an analysis of how it influences the evolutionary dynamics of disease-causing variants has not yet been explored. We develop a simple population genetic model of disease susceptibility and evaluate it using a biologically plausible empirically based distribution of fitness effects among contributing mutations. The model predicts that alleles under sex-differential selection, including sexually antagonistic alleles, will disproportionately contribute to genetic variation for disease predisposition, thereby generating substantial sexual dimorphism in the genetic architecture of complex (polygenic) diseases. This is because such alleles evolve into higher population frequencies for a given effect size, relative to alleles experiencing equally strong purifying selection in both sexes. Our results provide a theoretical justification for expecting a sexually dimorphic genetic basis for variation in complex traits such as disease. Moreover, they suggest that such dimorphism is interesting - not merely something to control for - because it reflects the action of natural selection in molding the evolution of common disease phenotypes.
Human monoclonal IgM with autoantibody activity against two gangliosides (GM1 and GD1b) in a patient with motor neuron syndrome.

PubMed

Jauberteau, M O; Gualde, N; Preud'Homme, J L; Rigaud, M; Gil, R; Vallat, J M; Baumann, N

1990-05-01

Small amounts of oligoclonal immunoglobulins were detected by Western blotting in the serum from a patient with motor neuron syndrome. The prominent one, a monoclonal IgM lambda, reacted strongly with the gangliosides GM1 and GD1b and more weakly with asialo GM1, as shown by immunoenzymatic staining of thin-layer chromatograms of gangliosides, ELISA on purified glycolipid coats and immunoadsorption with purified GM1. Affinity-chromatography with purified GM1 resulted in the purification of monoclonal IgM lambda. This purified IgM and its Fab fragments showed the same pattern of reactivity with gangliosides as that observed with whole serum. Such monoclonal IgM could be responsible for motor neuron diseases in some patients with overt or barely detectable monoclonal gammopathies.
Lactosylamidine-based affinity purification for cellulolytic enzymes EG I and CBH I from Hypocrea jecorina and their properties.

PubMed

Ogata, Makoto; Kameshima, Yumiko; Hattori, Takeshi; Michishita, Kousuke; Suzuki, Tomohiro; Kawagishi, Hirokazu; Totani, Kazuhide; Hiratake, Jun; Usui, Taichi

2010-12-10

Selective adsorption and separation of β-glucosidase, endo-acting endo-β-(1→4)-glucanase I (EG I), and exo-acting cellobiohydrolase I (CBH I) were achieved by affinity chromatography with β-lactosylamidine as ligand. A crude cellulase preparation from Hypocrea jecorina served as the source of enzyme. When crude cellulase was applied to the lactosylamidine-based affinity column, β-glucosidase appeared in the unbound fraction. By contrast, EG I and CBH I were retained on the column and then separated from each other by appropriately adjusting the elution conditions. The relative affinities of the enzymes, based on their column elution conditions, were strongly dependent on the ligand. The highly purified EG I and CBH I, obtained by affinity chromatography, were further purified by Mono P and DEAE chromatography, respectively. EG I and CBH I cleave only at the phenolic bond in p-nitrophenyl glycosides with lactose and N-acetyllactosamine (LacNAc). By contrast, both scissile bonds in p-nitrophenyl glycosides with cellobiose were subject to hydrolysis although with important differences in their kinetic parameters. Copyright © 2010 Elsevier Ltd. All rights reserved.
Construction of a chimeric thermostable pyrophosphatase to facilitate its purification and immobilization by using the choline-binding tag.

PubMed

Moldes, Cristina; García, José L; García, Pedro

2004-08-01

The thermophilic inorganic pyrophosphatase (Pyr) from Thermus thermophilus has been produced in Escherichia coli fused to the C terminus of the choline-binding tag (ChB tag) derived from the choline-binding domain (ChBD) of pneumococcal LytA autolysin. The chimeric ChBD-Pyr protein retains its thermostable activity and can be purified in a single step by DEAE-cellulose affinity chromatography. Pyr can be further released from the ChBD by thrombin, using the specific protease recognition site incorporated in the C terminus of this tag. Remarkably, the ChB tag provides a selective and very strong thermostable noncovalent immobilization of ChBD-Pyr in the DEAE-cellulose matrix. The binding of choline or choline analogues, such as DEAE, confers a high thermal stability to this tag; therefore, the immobilized chimeric enzyme can be assayed at high temperature without protein leakage, demonstrating the usefulness of the ChB tag for noncovalent immobilization of thermophilic proteins. Moreover, ChBD-Pyr can be purified and immobilized in a single step on commercial DEAE-cellulose paper. The affinity of the ChB tag for this versatile solid support can be very helpful in developing many biotechnological applications.
A general method for the purification of synthetic oligodeoxyribonucleotides containing strong secondary structure by reversed-phase high-performance liquid chromatography on PRP-1 resin.

PubMed

Germann, M W; Pon, R T; van de Sande, J H

1987-09-01

Synthetic 5'-dimethoxytritylated oligodeoxyribonucleotides, which contained strong secondary structure, were satisfactorily denatured and purified by reversed-phase HPLC on PRP-1 columns when strongly alkaline conditions (0.05 M NaOH) were employed. This procedure was suitable for the purification of hairpin structures, e.g., d(CG)nT4(CG)n (n = 4, 5, 6), and oligo(dG) sequences, e.g., d(G)24, as well as oligodeoxyribonucleotide probes which contained degenerate base sites. Oligodeoxyribonucleotides as long as 50 bases in length were purified. Recovery of injected oligonucleotides was typically 90% or better. The high capacity of the PRP-1 resin also allowed purification to be performed on a preparative scale (2-8 mg per injection). Enzymatic degradation and HPLC analysis indicated that no modification of the heterocyclic bases occurred under the alkaline conditions described.
Facile Isolation of Adsorbent-Free Long and Highly-Pure Chirality-Selected Semiconducting Single-Walled Carbon Nanotubes Using A Hydrogen-bonding Supramolecular Polymer.

PubMed

Toshimitsu, Fumiyuki; Nakashima, Naotoshi

2015-12-14

The ideal form of semiconducting-single-walled carbon nanotubes (sem-SWNTs) for science and technology is long, defect-free, chirality pure and chemically pure isolated narrow diameter tubes. While various techniques to solubilize and purify sem-SWNTs have been developed, many of them targeted only the chiral- or chemically-purity while sacrificing the sem-SWNT intrinsic structural identities by applying strong ultra-sonication and/or chemical modifications. Toward the ultimate purification of the sem-SWNTs, here we report a mild-conditioned extraction of the sem-SWNTs using removable supramolecular hydrogen-bonding polymers (HBPs) that are composed of dicarboxylic- or diaminopyridyl-fluorenes with ~70%-(8,6)SWNT selective extraction. Replacing conventional strong sonication techniques by a simple shaking using HPBs was found to provide long sem-SWNTs (>2.0 μm) with a very high D/G ratio, which was determined by atomic force microscopy observations. The HBPs were readily removed from the nanotube surfaces by an outer stimulus, such as a change in the solvent polarities, to provide chemically pure (8,6)-enriched sem-SWNTs. We also describe molecular mechanics calculations to propose possible structures for the HBP-wrapped sem-SWNTs, furthermore, the mechanism of the chiral selectivity for the sorted sem-SWNTs is well explained by the relationship between the molecular surface area and mass of the HBP/SWNT composites.
Selfish drive can trump function when animal mitochondrial genomes compete.

PubMed

Ma, Hansong; O'Farrell, Patrick H

2016-07-01

Mitochondrial genomes compete for transmission from mother to progeny. We explored this competition by introducing a second genome into Drosophila melanogaster to follow transmission. Competitions between closely related genomes favored those functional in electron transport, resulting in a host-beneficial purifying selection. In contrast, matchups between distantly related genomes often favored those with negligible, negative or lethal consequences, indicating selfish selection. Exhibiting powerful selfish selection, a genome carrying a detrimental mutation displaced a complementing genome, leading to population death after several generations. In a different pairing, opposing selfish and purifying selection counterbalanced to give stable transmission of two genomes. Sequencing of recombinant mitochondrial genomes showed that the noncoding region, containing origins of replication, governs selfish transmission. Uniparental inheritance prevents encounters between distantly related genomes. Nonetheless, in each maternal lineage, constant competition among sibling genomes selects for super-replicators. We suggest that this relentless competition drives positive selection, promoting change in the sequences influencing transmission.
Selfish drive can trump function when animal mitochondrial genomes compete

PubMed Central

Ma, Hansong; O’Farrell, Patrick H.

2016-01-01

Mitochondrial genomes compete for transmission from mother to progeny. We explored this competition by introducing a second genome into Drosophila melanogaster to follow transmission. Competitions between closely related genomes favored those functional in electron transport, resulting in a host-beneficial purifying selection1. Contrastingly, matchups between distant genomes often favored those with negligible, negative or lethal consequences, indicating selfish selection. Exhibiting powerful selfish selection, a genome carrying a detrimental mutation displaced a complementing genome leading to population death after several generations. In a different pairing, opposing selfish and purifying selection counterbalanced to give stable transmission of two genomes. Sequencing of recombinant mitochondrial genomes revealed that the non-coding region, containing origins of replication, governs selfish transmission. Uniparental inheritance prevents encounters between distantly related genomes. Nonetheless, within each maternal lineage, constant competition among sibling genomes selects for super-replicators. We suggest that this relentless competition drives positive selection promoting change in the sequences influencing transmission. PMID:27270106
Landscape genomics: natural selection drives the evolution of mitogenome in penguins.

PubMed

Ramos, Barbara; González-Acuña, Daniel; Loyola, David E; Johnson, Warren E; Parker, Patricia G; Massaro, Melanie; Dantas, Gisele P M; Miranda, Marcelo D; Vianna, Juliana A

2018-01-16

Mitochondria play a key role in the balance of energy and heat production, and therefore the mitochondrial genome is under natural selection by environmental temperature and food availability, since starvation can generate more efficient coupling of energy production. However, selection over mitochondrial DNA (mtDNA) genes has usually been evaluated at the population level. We sequenced by NGS 12 mitogenomes and with four published genomes, assessed genetic variation in ten penguin species distributed from the equator to Antarctica. Signatures of selection of 13 mitochondrial protein-coding genes were evaluated by comparing among species within and among genera (Spheniscus, Pygoscelis, Eudyptula, Eudyptes and Aptenodytes). The genetic data were correlated with environmental data obtained through remote sensing (sea surface temperature [SST], chlorophyll levels [Chl] and a combination of SST and Chl [COM]) through the distribution of these species. We identified the complete mtDNA genomes of several penguin species, including ND6 and 8 tRNAs on the light strand and 12 protein coding genes, 14 tRNAs and two rRNAs positioned on the heavy strand. The highest diversity was found in NADH dehydrogenase genes and the lowest in COX genes. The lowest evolutionary divergence among species was between Humboldt (Spheniscus humboldti) and Galapagos (S. mendiculus) penguins (0.004), while the highest was observed between little penguin (Eudyptula minor) and Adélie penguin (Pygoscelis adeliae) (0.097). We identified a signature of purifying selection (Ka/Ks < 1) across the mitochondrial genome, which is consistent with the hypothesis that purifying selection is constraining mitogenome evolution to maintain Oxidative phosphorylation (OXPHOS) proteins and functionality. Pairwise species maximum-likelihood analyses of selection at codon sites suggest positive selection has occurred on ATP8 (Fixed-Effects Likelihood, FEL) and ND4 (Single Likelihood Ancestral Counting, SLAC) in all penguins. In contrast, COX1 had a signature of strong negative selection. ND4 Ka/Ks ratios were highly correlated with SST (Mantel, p-value: 0.0001; GLM, p-value: 0.00001) and thus may be related to climate adaptation throughout penguin speciation. These results identify mtDNA candidate genes under selection which could be involved in broad-scale adaptations of penguins to their environment. Such knowledge may be particularly useful for developing predictive models of how these species may respond to severe climatic changes in the future.
Molecular evolution of the endosperm starch synthesis pathway genes in rice (Oryza sativa L.) and its wild ancestor, O. rufipogon L.

PubMed

Yu, Guoqin; Olsen, Kenneth M; Schaal, Barbara A

2011-01-01

The evolution of metabolic pathways is a fundamental but poorly understood aspect of evolutionary change. One approach for understanding the complexity of pathway evolution is to examine the molecular evolution of genes that together comprise an integrated metabolic pathway. The rice endosperm starch biosynthetic pathway is one of the most thoroughly characterized metabolic pathways in plants, and starch is a trait that has evolved in response to strong selection during rice domestication. In this study, we have examined six key genes (AGPL2, AGPS2b, SSIIa, SBEIIb, GBSSI, ISA1) in the rice endosperm starch biosynthesis pathway to investigate the evolution of these genes before and after rice domestication. Genome-wide sequence tagged sites data were used as a neutral reference to overcome the problems of detecting selection in species with complex demographic histories such as rice. Five variety groups of Oryza sativa (aus, indica, tropical japonica, temperate japonica, aromatic) and its wild ancestor (O. rufipogon) were sampled. Our results showed evidence of purifying selection at AGPL2 in O. rufipogon and strong evidence of positive selection at GBSSI in temperate japonica and tropical japonica varieties and at GBSSI and SBEIIb in aromatic varieties. All the other genes showed a pattern consistent with neutral evolution in both cultivated rice and its wild ancestor. These results indicate the important role of positive selection in the evolution of starch genes during rice domestication. We discuss the role of SBEIIb and GBSSI in the evolution of starch quality during rice domestication and the power and limitation of detecting selection using genome-wide data as a neutral reference.
Comparative analysis of complete mitochondrial genomes suggests that relaxed purifying selection is driving high nonsynonymous evolutionary rate of the NADH2 gene in whitefish (Coregonus ssp.).

PubMed

Jacobsen, Magnus W; da Fonseca, Rute R; Bernatchez, Louis; Hansen, Michael M

2016-02-01

Several studies have recently reported evidence for positive selection acting on the mitochondrial genome (mitogenome), emphasizing its potential role in adaptive divergence and speciation. In this study we searched 107 full mitogenomes of recently diverged species and lineages of whitefish (Coregonus ssp.) for signals of positive selection. These salmonids show several distinct morphological and ecological differences that may be associated with energetics and therefore potentially positive selection at the mitogenome level. We found that purifying selection and genetic drift were the predominant evolutionary forces acting on the analyzed mitogenomes. However, the NADH dehydrogenase 2 gene (ND2) showed a highly elevated dN/dS ratio compared to the other mitochondrial genes, which was significantly higher in whitefish compared to other salmonids. We therefore further examined nonsynonymous evolution in ND2 by (i) mapping amino acid changes to a protein model structure which showed that they were located away from key functional residues of the protein, (ii) locating them in the sequences of other species of fish (Salmonidae, Anguillidae, Scombridae and Percidae) only to find pronounced overlap of nonsynonymous regions. We thus conclude that relaxed purifying selection is driving the evolution of ND2 by affecting mostly regions that have lower functional relevance. Copyright © 2015 Elsevier Inc. All rights reserved.
ANIONIC EXCHANGE PROCESS FOR THE RECOVERY OF URANIUM AND VANADIUM FROM CARBONATE SOLUTIONS

DOEpatents

Bailes, R.H.; Ellis, D.A.; Long, R.S.

1958-12-16

Uranium and vanadium can be economically purified and recovered from non- salt roast carbonate leach liquors by adsorption on a strongly basic anionic exchange resin and subsequent selective elution by one of three alternative methods. Method 1 comprises selectively eluting uranium from the resin with an ammonium sulfate solution followed by eluting vanadium from the resin with either 5 M NaCl, saturated (NH/sub 4/)/sub 2/CO/sub 3/, saturated NaHCO/sub 3/, 1 M NaOH, or saturated S0/sub 2/ solutions. Method II comprises selectively eluting vanadium from the resin with either concentrated NaCl or S0/sub 2/ solutions subsequent to pretreatment of the column with either S0/sub 2/ gas, 1 N HCl, or 0.1 N H/sub 2/8O/sub 4/ followed by eluting uranium from the resin with solutions containing 0.9 M NH/sub 4/Cl or NaCl and 0.1 Cl. Method III comprises flowing the carbonate leac solutlon through a first column of a strongly basic anlonlc exchange resin untll vanadium breakthrough occurs, so that the effluent solution is enriched ln uranium content and the vanadium is chiefly retalned by the resln, absorbing the uranlum from the enriched effluent solution on a second column of a strongly basic anionic exchange resin, pretreating the first column with either 0.1 N HCl, 0.1 H/sub 2/SO/sub 4/, C0/sub 2/ gas, or ammonium sulfate, selectively eluting the vanadlum from the column with saturated S0/sub 2/ solution, pretreatlng the second column with either 0.1 N HCl or S0/sub 2/ gas, selectively eluting residual vanadium from the column with saturated S0/sub 2/ solution, and then eluting the uranium from the column with either 0.1 N HCl and 1 N NaCl orO.l N HCl and 1 N NH/sub 4/Cl.

Mosquitoes Transmit Unique West Nile Virus Populations during Each Feeding Episode.

PubMed

Grubaugh, Nathan D; Fauver, Joseph R; Rückert, Claudia; Weger-Lucarelli, James; Garcia-Luna, Selene; Murrieta, Reyes A; Gendernalik, Alex; Smith, Darci R; Brackney, Doug E; Ebel, Gregory D

2017-04-25

Arthropod-borne viruses (arboviruses), such as Zika virus, chikungunya virus, and West Nile virus (WNV), pose continuous threats to emerge and cause large epidemics. Often, these events are associated with novel virus variants optimized for local transmission that first arise as minorities within a host. Thus, the conditions that regulate the frequency of intrahost variants are important determinants of emergence. Here, we describe the dynamics of WNV genetic diversity during its transmission cycle. By temporally sampling saliva from individual mosquitoes, we demonstrate that virus populations expectorated by mosquitoes are highly diverse and unique to each feeding episode. After transmission to birds, however, most genetic diversity is removed by strong purifying selection. Further, transmission of potentially mosquito-adaptive WNV variants is strongly influenced by genetic drift in mosquitoes. These results highlight the complex evolutionary forces a novel virus variant must overcome to alter infection phenotypes at the population level. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.
Human monoclonal IgM with autoantibody activity against two gangliosides (GM1 and GD1b) in a patient with motor neuron syndrome.

PubMed Central

Jauberteau, M O; Gualde, N; Preud'Homme, J L; Rigaud, M; Gil, R; Vallat, J M; Baumann, N

1990-01-01

Small amounts of oligoclonal immunoglobulins were detected by Western blotting in the serum from a patient with motor neuron syndrome. The prominent one, a monoclonal IgM lambda, reacted strongly with the gangliosides GM1 and GD1b and more weakly with asialo GM1, as shown by immunoenzymatic staining of thin-layer chromatograms of gangliosides, ELISA on purified glycolipid coats and immunoadsorption with purified GM1. Affinity-chromatography with purified GM1 resulted in the purification of monoclonal IgM lambda. This purified IgM and its Fab fragments showed the same pattern of reactivity with gangliosides as that observed with whole serum. Such monoclonal IgM could be responsible for motor neuron diseases in some patients with overt or barely detectable monoclonal gammopathies. Images Fig. 2 Fig. 3 PMID:2357844
Characterization of poly(L-lactide)-degrading enzyme produced by thermophilic filamentous bacteria Laceyella sacchari LP175.

PubMed

Hanphakphoom, Srisuda; Maneewong, Narisara; Sukkhum, Sukhumaporn; Tokuyama, Shinji; Kitpreechavanich, Vichien

2014-01-01

Eleven strains of poly(L-lactide) (PLLA)-degrading thermophilic bacteria were isolated from forest soils and selected based on clear zone formation on an emulsified PLLA agar plate at 50°C. Among the isolates, strain LP175 showed the highest PLLA-degrading ability. It was closely related to Laceyella sacchari, with 99.9% similarity based on the 16S rRNA gene sequence. The PLLA-degrading enzyme produced by the strain was purified to homogeneity by 48.1% yield and specific activity of 328 U·mg-protein-1 with a 15.3-fold purity increase. The purified enzyme was strongly active against specific substrates such as casein and gelatin and weakly active against Suc-(Ala)₃-pNA. Optimum enzyme activity was exhibited at a temperature of 60°C with thermal stability up to 50°C and a pH of 9.0 with pH stability in a range of 8.5-10.5. Molecular weight of the enzyme was approximately 28.0 kDa, as determined by gel filtration and SDS-PAGE. The inhibitors phenylmethylsulfonyl fluoride (PMSF), ethylenediaminetetraacetate (EDTA), and ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA) strongly inhibited enzyme activity, but the activity was not inhibited by 1 mM 1,10-phenanthroline (1,10-phen). The N-terminal amino acid sequences had 100% homology with thermostable serine protease (thermitase) from Thermoactinomyces vulgaris. The results obtained suggest that the PLLA-degrading enzyme produced by L. sacchari strain LP175 is serine protease.
Differences in selection drive olfactory receptor genes in different directions in dogs and wolf.

PubMed

Chen, Rui; Irwin, David M; Zhang, Ya-Ping

2012-11-01

The olfactory receptor (OR) gene family is the largest gene family found in mammalian genomes. It is known to evolve through a birth-and-death process. Here, we characterized the sequences of 16 segregating OR pseudogenes in the samples of the wolf and the Chinese village dog (CVD) and compared them with the sequences from dogs of different breeds. Our results show that the segregating OR pseudogenes in breed dogs are under strong purifying selection, while evolving neutrally in the CVD, and show a more complicated pattern in the wolf. In the wolf, we found a trend to remove deleterious polymorphisms and accumulate nondeleterious polymorphisms. On the basis of protein structure of the ORs, we found that the distribution of different types of polymorphisms (synonymous, nonsynonymous, tolerated, and untolerated) varied greatly between the wolf and the breed dogs. In summary, our results suggest that different forms of selection have acted on the segregating OR pseudogenes in the CVD since domestication, breed dogs after breed formation, and ancestral wolf population, which has driven the evolution of these genes in different directions.
Genome-wide sequence variations between wild and cultivated tomato species revisited by whole genome sequence mapping.

PubMed

Sahu, Kamlesh Kumar; Chattopadhyay, Debasis

2017-06-02

Cultivated tomato (Solanum lycopersicum L.) is the second most important vegetable crop after potato and a member of thirteen interfertile species of Solanum genus. Domestication and continuous selection for desirable traits made cultivated tomato species susceptible to many stresses as compared to the wild species. In this study, we analyzed and compared the genomes of wild and cultivated tomato accessions to identify the genomic regions that encountered changes during domestication. Analysis was based on SNP and InDel mining of twentynine accessions of twelve wild tomato species and forty accessions of cultivated tomato. Percentage of common SNPs among the accessions within a species corresponded with the reproductive behavior of the species. SNP profiles of the wild tomato species within a phylogenetic subsection varied with their geographical distribution. Interestingly, the ratio of genic SNP to total SNPs increased with phylogenetic distance of the wild tomato species from the domesticated species, suggesting that variations in gene-coding region play a major role in speciation. We retrieved 2439 physical positions in 1594 genes including 32 resistance related genes where all the wild accessions possessed a common wild variant allele different from all the cultivated accessions studied. Tajima's D analysis predicted a very strong purifying selection associated with domestication in nearly 1% of its genome, half of which is contributed by chromosome 11. This genomic region with a low Tajima's D value hosts a variety of genes associated with important agronomic trait such as, fruit size, tiller number and wax deposition. Our analysis revealed a broad-spectrum genetic base in wild tomato species and erosion of that in cultivated tomato due to recurrent selection for agronomically important traits. Identification of the common wild variant alleles and the genomic regions undergoing purifying selection during cultivation would facilitate future breeding program by introgression from wild species.
Purified glycosaminoglycans from cooked haddock may enhance Fe uptake via endocytosis in a Caco-2 cell culture model

USDA-ARS?s Scientific Manuscript database

This study aims to understand the enhancing effect of glycosaminoglycans (GAGs), such as chondroitin/dermatan structures, on Fe uptake to Caco-2 cells. High sulfated GAGs were selectively purified from cooked haddock. An in vitro digestion/Caco-2 cell culture model was used to evaluate Fe uptake (ce...
Contrasted evolutionary histories of two Toll-like receptors (Tlr4 and Tlr7) in wild rodents (MURINAE)

PubMed Central

2013-01-01

Background In vertebrates, it has been repeatedly demonstrated that genes encoding proteins involved in pathogen-recognition by adaptive immunity (e.g. MHC) are subject to intensive diversifying selection. On the other hand, the role and the type of selection processes shaping the evolution of innate-immunity genes are currently far less clear. In this study we analysed the natural variation and the evolutionary processes acting on two genes involved in the innate-immunity recognition of Microbe-Associated Molecular Patterns (MAMPs). Results We sequenced genes encoding Toll-like receptor 4 (Tlr4) and 7 (Tlr7), two of the key bacterial- and viral-sensing receptors of innate immunity, across 23 species within the subfamily Murinae. Although we have shown that the phylogeny of both Tlr genes is largely congruent with the phylogeny of rodents based on a comparably sized non-immune sequence dataset, we also identified several potentially important discrepancies. The sequence analyses revealed that major parts of both Tlrs are evolving under strong purifying selection, likely due to functional constraints. Yet, also several signatures of positive selection have been found in both genes, with more intense signal in the bacterial-sensing Tlr4 than in the viral-sensing Tlr7. 92% and 100% of sites evolving under positive selection in Tlr4 and Tlr7, respectively, were located in the extracellular domain. Directly in the Ligand-Binding Region (LBR) of TLR4 we identified two rapidly evolving amino acid residues and one site under positive selection, all three likely involved in species-specific recognition of lipopolysaccharide of gram-negative bacteria. In contrast, all putative sites of LBRTLR7 involved in the detection of viral nucleic acids were highly conserved across rodents. Interspecific differences in the predicted 3D-structure of the LBR of both Tlrs were not related to phylogenetic history, while analyses of protein charges clearly discriminated Rattini and Murini clades. Conclusions In consequence of the constraints given by the receptor protein function purifying selection has been a dominant force in evolution of Tlrs. Nevertheless, our results show that episodic diversifying parasite-mediated selection has shaped the present species-specific variability in rodent Tlrs. The intensity of diversifying selection was higher in Tlr4 than in Tlr7, presumably due to structural properties of their ligands. PMID:24028551
Purified ryanodine receptor from rabbit skeletal muscle is the calcium- release channel of sarcoplasmic reticulum

PubMed Central

1988-01-01

The ryanodine receptor of rabbit skeletal muscle sarcoplasmic reticulum was purified as a single 450,000-dalton polypeptide from CHAPS- solubilized triads using immunoaffinity chromatography. The purified receptor had a [3H]ryanodine-binding capacity (Bmax) of 490 pmol/mg and a binding affinity (Kd) of 7.0 nM. Using planar bilayer recording techniques, we show that the purified receptor forms cationic channels selective for divalent ions. Ryanodine receptor channels were identical to the Ca-release channels described in native sarcoplasmic reticulum using the same techniques. In the present work, four criteria were used to establish this identity: (a) activation of channels by micromolar Ca and millimolar ATP and inhibition by micromolar ruthenium red, (b) a main channel conductance of 110 +/- 10 pS in 54 mM trans Ca, (c) a long- term open state of lower unitary conductance induced by ryanodine concentrations as low as 20 nM, and (d) a permeability ratio PCa/PTris approximately equal to 14. In addition, we show that the purified ryanodine receptor channel displays a saturable conductance in both monovalent and divalent cation solutions (gamma max for K and Ca = 1 nS and 172 pS, respectively). In the absence of Ca, channels had a broad selectivity for monovalent cations, but in the presence of Ca, they were selectively permeable to Ca against K by a permeability ratio PCa/PK approximately equal to 6. Receptor channels displayed several equivalent conductance levels, which suggest an oligomeric pore structure. We conclude that the 450,000-dalton polypeptide ryanodine receptor is the Ca-release channel of the sarcoplasmic reticulum and is the target site of ruthenium red and ryanodine. PMID:2459298
Seagrass as a potential source of natural antioxidant and anti-inflammatory agents.

PubMed

Yuvaraj, N; Kanmani, P; Satishkumar, R; Paari, A; Pattukumar, V; Arul, V

2012-04-01

Halophila spp. is a strong medicine against malaria and skin diseases and is found to be very effective in early stages of leprosy. Seagrasses are nutraceutical in nature and therefore of importance as food supplements. The antibacterial, antioxidant, and anti-inflammatory activities of Halophila ovalis R. Br. Hooke (Hydrocharitaceae) methanol extract were investigated and the chemical constituents of purified fractions were analyzed. Plant materials were collected from Pondicherry coastal line, and antimicrobial screening of crude extract, and purified fractions was carried out by the disc diffusion method and the minimum inhibitory concentration (MICs) of the purified fractions and reference antibiotics were determined by microdilution method. Antioxidant and anti-inflammatory activities were investigated in vitro. Chemical constituents of purified fractions V and VI were analyzed by gas chromatography-mass spectrometry (GC-MS), and the phytochemicals were quantitatively determined. Methanol extract inhibited the growth of Bacillus cereus at a minimum inhibitory concentration of 50 µg/mL and other Gram-negative pathogens at 75 µg/ml, except Vibrio vulnificus. Reducing power and total antioxidant level increased with increasing extract concentration. H. ovalis exhibited strong scavenging activity on 2,2-diphenyl-1-picrylhydrazyl (DPPH) and superoxide radicals at IC(50) of 0.13 and 0.65 mg/mL, respectively. Methanol extract of H. ovalis showed noticeable anti-inflammatory activity at IC(50) of 78.72 µg/mL. The GC-MS analysis of H. ovalis revealed the presence of triacylglycerols as major components in purified fractions. Quantitative analysis of phytochemicals revealed that phenols are rich in seagrass H. ovalis. These findings demonstrated that the methanol extract of H. ovalis exhibited appreciable antibacterial, noticeable antioxidant, and anti-inflammatory activities, and thus could be use as a potential source for natural health products.
Development of a new measurement system to detect selectively volatile organic compounds derived from the human body.

PubMed

Kanou, S; Nagaoka, T; Kobayashi, N; Kurahashi, M; Takeda, S; Aoki, T; Tsuji, T; Urano, T; Abe, T; Magatani, K

2013-01-01

A new concept expired gas measurement system used double cold-trap method was developed. The system could detect selectively volatile organic compound (VOC) derived from the human body. The gas chromatography (GC) profiles of healthy volunteer's expired gas collected by our system were analyzed. As a result, 60 VOCs were detected from the healthy volunteer's expired gas. We examined 14 VOCs among them further, which could be converted to the concentration from the GC profiles. The concentration of almost VOCs decreased when the subjects inspired purified air compared with the atmosphere. On the other hand, isoprene was almost the same. It was strongly suggested that these VOCs were derived from the human body because the concentration of these VOCs in the atmosphere were nearly zero. Expired gas of two sleep apnea syndrome (SAS) patients were analyzed as preliminary study. As a result of the study, the concentration of some VOCs contained in the expired gas of the SAS patients showed higher value than a healthy controls.
Evolutionary dynamics of Newcastle disease virus

USGS Publications Warehouse

Miller, P.J.; Kim, L.M.; Ip, Hon S.; Afonso, C.L.

2009-01-01

A comprehensive dataset of NDV genome sequences was evaluated using bioinformatics to characterize the evolutionary forces affecting NDV genomes. Despite evidence of recombination in most genes, only one event in the fusion gene of genotype V viruses produced evolutionarily viable progenies. The codon-associated rate of change for the six NDV proteins revealed that the highest rate of change occurred at the fusion protein. All proteins were under strong purifying (negative) selection; the fusion protein displayed the highest number of amino acids under positive selection. Regardless of the phylogenetic grouping or the level of virulence, the cleavage site motif was highly conserved implying that mutations at this site that result in changes of virulence may not be favored. The coding sequence of the fusion gene and the genomes of viruses from wild birds displayed higher yearly rates of change in virulent viruses than in viruses of low virulence, suggesting that an increase in virulence may accelerate the rate of NDV evolution. ?? 2009 Elsevier Inc.
INVESTIGATIONS ON THE ANTIGENICITY OF SNAKE VENOMS

DTIC Science & Technology

The chemical composition of viperotoxin, the neurotoxic protein isolated from the venom of Vipera palestinae has been determined. Viperotoxin is...carboxy-terminal position of the viperotoxin chain. Vipera palestinae hemorrhagin has been purified and isolated. Further tests have established that...active sites, or selective blocking of a part of one active center having two distinct biological activities. Purified neurotoxin of V. palestinae
Methods for purifying carbon materials

DOEpatents

Dailly, Anne [Pasadena, CA; Ahn, Channing [Pasadena, CA; Yazami, Rachid [Los Angeles, CA; Fultz, Brent T [Pasadena, CA

2009-05-26

Methods of purifying samples are provided that are capable of removing carbonaceous and noncarbonaceous impurities from a sample containing a carbon material having a selected structure. Purification methods are provided for removing residual metal catalyst particles enclosed in multilayer carbonaceous impurities in samples generate by catalytic synthesis methods. Purification methods are provided wherein carbonaceous impurities in a sample are at least partially exfoliated, thereby facilitating subsequent removal of carbonaceous and noncarbonaceous impurities from the sample. Methods of purifying carbon nanotube-containing samples are provided wherein an intercalant is added to the sample and subsequently reacted with an exfoliation initiator to achieve exfoliation of carbonaceous impurities.
Comparative Genomics Provide Insights into Evolution of Trichoderma Nutrition Style

PubMed Central

Xie, Bin-Bin; Qin, Qi-Long; Shi, Mei; Chen, Lei-Lei; Shu, Yan-Li; Luo, Yan; Wang, Xiao-Wei; Rong, Jin-Cheng; Gong, Zhi-Ting; Li, Dan; Sun, Cai-Yun; Liu, Gui-Ming; Dong, Xiao-Wei; Pang, Xiu-Hua; Huang, Feng; Liu, Weifeng; Chen, Xiu-Lan; Zhou, Bai-Cheng; Zhang, Yu-Zhong; Song, Xiao-Yan

2014-01-01

Saprotrophy on plant biomass is a recently developed nutrition strategy for Trichoderma. However, the physiology and evolution of this new nutrition strategy is still elusive. We report the deep sequencing and analysis of the genome of Trichoderma longibrachiatum, an efficient cellulase producer. The 31.7-Mb genome, smallest among the sequenced Trichoderma species, encodes fewer nutrition-related genes than saprotrophic T. reesei (Tr), including glycoside hydrolases and nonribosomal peptide synthetase–polyketide synthase. Homology and phylogenetic analyses suggest that a large number of nutrition-related genes, including GH18 chitinases, β-1,3/1,6-glucanases, cellulolytic enzymes, and hemicellulolytic enzymes, were lost in the common ancestor of T. longibrachiatum (Tl) and Tr. dN/dS (ω) calculation indicates that all the nutrition-related genes analyzed are under purifying selection. Cellulolytic enzymes, the key enzymes for saprotrophy on plant biomass, are under stronger purifying selection pressure in Tl and Tr than in mycoparasitic species, suggesting that development of the nutrition strategy of saprotrophy on plant biomass has increased the selection pressure. In addition, aspartic proteases, serine proteases, and metalloproteases are subject to stronger purifying selection pressure in Tl and Tr, suggesting that these enzymes may also play important roles in the nutrition. This study provides insights into the physiology and evolution of the nutrition strategy of Trichoderma. PMID:24482532
Comparative genomics provide insights into evolution of trichoderma nutrition style.

PubMed

Xie, Bin-Bin; Qin, Qi-Long; Shi, Mei; Chen, Lei-Lei; Shu, Yan-Li; Luo, Yan; Wang, Xiao-Wei; Rong, Jin-Cheng; Gong, Zhi-Ting; Li, Dan; Sun, Cai-Yun; Liu, Gui-Ming; Dong, Xiao-Wei; Pang, Xiu-Hua; Huang, Feng; Liu, Weifeng; Chen, Xiu-Lan; Zhou, Bai-Cheng; Zhang, Yu-Zhong; Song, Xiao-Yan

2014-02-01

Saprotrophy on plant biomass is a recently developed nutrition strategy for Trichoderma. However, the physiology and evolution of this new nutrition strategy is still elusive. We report the deep sequencing and analysis of the genome of Trichoderma longibrachiatum, an efficient cellulase producer. The 31.7-Mb genome, smallest among the sequenced Trichoderma species, encodes fewer nutrition-related genes than saprotrophic T. reesei (Tr), including glycoside hydrolases and nonribosomal peptide synthetase-polyketide synthase. Homology and phylogenetic analyses suggest that a large number of nutrition-related genes, including GH18 chitinases, β-1,3/1,6-glucanases, cellulolytic enzymes, and hemicellulolytic enzymes, were lost in the common ancestor of T. longibrachiatum (Tl) and Tr. dN/dS (ω) calculation indicates that all the nutrition-related genes analyzed are under purifying selection. Cellulolytic enzymes, the key enzymes for saprotrophy on plant biomass, are under stronger purifying selection pressure in Tl and Tr than in mycoparasitic species, suggesting that development of the nutrition strategy of saprotrophy on plant biomass has increased the selection pressure. In addition, aspartic proteases, serine proteases, and metalloproteases are subject to stronger purifying selection pressure in Tl and Tr, suggesting that these enzymes may also play important roles in the nutrition. This study provides insights into the physiology and evolution of the nutrition strategy of Trichoderma.
Mitogenome Sequencing in the Genus Camelus Reveals Evidence for Purifying Selection and Long-term Divergence between Wild and Domestic Bactrian Camels.

PubMed

Mohandesan, Elmira; Fitak, Robert R; Corander, Jukka; Yadamsuren, Adiya; Chuluunbat, Battsetseg; Abdelhadi, Omer; Raziq, Abdul; Nagy, Peter; Stalder, Gabrielle; Walzer, Chris; Faye, Bernard; Burger, Pamela A

2017-08-30

The genus Camelus is an interesting model to study adaptive evolution in the mitochondrial genome, as the three extant Old World camel species inhabit hot and low-altitude as well as cold and high-altitude deserts. We sequenced 24 camel mitogenomes and combined them with three previously published sequences to study the role of natural selection under different environmental pressure, and to advance our understanding of the evolutionary history of the genus Camelus. We confirmed the heterogeneity of divergence across different components of the electron transport system. Lineage-specific analysis of mitochondrial protein evolution revealed a significant effect of purifying selection in the concatenated protein-coding genes in domestic Bactrian camels. The estimated dN/dS < 1 in the concatenated protein-coding genes suggested purifying selection as driving force for shaping mitogenome diversity in camels. Additional analyses of the functional divergence in amino acid changes between species-specific lineages indicated fixed substitutions in various genes, with radical effects on the physicochemical properties of the protein products. The evolutionary time estimates revealed a divergence between domestic and wild Bactrian camels around 1.1 [0.58-1.8] million years ago (mya). This has major implications for the conservation and management of the critically endangered wild species, Camelus ferus.
Molecular Evolution of the Infrared Sensory Gene TRPA1 in Snakes and Implications for Functional Studies

PubMed Central

Jiang, Ke; Zhang, Peng

2011-01-01

TRPA1 is a calcium ion channel protein recently identified as the infrared receptor in pit organ-containing snakes. Therefore, understanding the molecular evolution of TRPA1 may help to illuminate the origin of “heat vision” in snakes and reveal the molecular mechanism of infrared sensitivity for TRPA1. To this end, we sequenced the infrared sensory gene TRPA1 in 24 snake species, representing nine snake families and multiple non-snake outgroups. We found that TRPA1 is under strong positive selection in the pit-bearing snakes studied, but not in other non-pit snakes and non-snake vertebrates. As a comparison, TRPV1, a gene closely related to TRPA1, was found to be under strong purifying selection in all the species studied, with no difference in the strength of selection between pit-bearing snakes and non-pit snakes. This finding demonstrates that the adaptive evolution of TRPA1 specifically occurred within the pit-bearing snakes and may be related to the functional modification for detecting infrared radiation. In addition, by comparing the TRPA1 protein sequences, we identified 11 amino acid sites that were diverged in pit-bearing snakes but conserved in non-pit snakes and other vertebrates, 21 sites that were diverged only within pit-vipers but conserved in the remaining snakes. These specific amino acid substitutions may be potentially functional important for infrared sensing. PMID:22163322
Developmental constraints shape the evolution of the nematode mid-developmental transition.

PubMed

Zalts, Harel; Yanai, Itai

2017-03-27

Evolutionary theory assumes that genetic variation is uniform and gradual in nature, yet morphological and gene expression studies have revealed that different life-stages exhibit distinct levels of cross-species conservation. In particular, a stage in mid-embryogenesis is highly conserved across species of the same phylum, suggesting that this stage is subject to developmental constraints, either by increased purifying selection or by a strong mutational bias. An alternative explanation, however, holds that the same 'hourglass' pattern of variation may result from increased positive selection at the earlier and later stages of development. To distinguish between these scenarios, we examined gene expression variation in a population of the nematode Caenorhabditis elegans using an experimental design that eliminated the influence of positive selection. By measuring gene expression for all genes throughout development in 20 strains, we found that variations were highly uneven throughout development, with a significant depletion during mid-embryogenesis. In particular, the family of homeodomain transcription factors, whose expression generally coincides with mid-embryogenesis, evolved under high constraint. Our data further show that genes responsible for the integration of germ layers during morphogenesis are the most constrained class of genes. Together, these results provide strong evidence for developmental constraints as the mechanism underlying the hourglass model of animal evolution. Understanding the pattern and mechanism of developmental constraints provides a framework to understand how evolutionary processes have interacted with embryogenesis and led to the diversity of animal life on Earth.
Reconstitution and functional comparison of purified GlpF and AqpZ, the glycerol and water channels from Escherichia coli.

PubMed

Borgnia, M J; Agre, P

2001-02-27

A large family of membrane channel proteins selective for transport of water (aquaporins) or water plus glycerol (aquaglyceroporins) has been found in diverse life forms. Escherichia coli has two members of this family-a water channel, AqpZ, and a glycerol facilitator, GlpF. Despite having similar primary amino acid sequences and predicted structures, the oligomeric state and solute selectivity of AqpZ and GlpF are disputed. Here we report biochemical and functional characterizations of affinity-purified GlpF and compare it to AqpZ. Histidine-tagged (His-GlpF) and hemagglutinin-tagged (HA-GlpF) polypeptides encoded by a bicistronic construct were expressed in bacteria. HA-GlpF and His-GlpF appear to form oligomers during Ni-nitrilotriacetate affinity purification. Sucrose gradient sedimentation analyses showed that the oligomeric state of octyl glucoside-solubilized GlpF varies: low ionic strength favors subunit dissociation, whereas Mg(2+) stabilizes tetrameric assembly. Reconstitution of affinity-purified GlpF into proteoliposomes increases glycerol permeability more than 100-fold and water permeability up to 10-fold compared with control liposomes. Glycerol and water permeability of GlpF both occur with low Arrhenius activation energies and are reversibly inhibited by HgCl(2). Our studies demonstrate that, unlike AqpZ, a water-selective stable tetramer, purified GlpF exists in multiple oligomeric forms under nondenaturing conditions and is highly permeable to glycerol but less well permeated by water.
Contemporary evolution of resistance at the major insecticide target site gene Ace-1 by mutation and copy number variation in the malaria mosquito Anopheles gambiae.

PubMed

Weetman, David; Mitchell, Sara N; Wilding, Craig S; Birks, Daniel P; Yawson, Alexander E; Essandoh, John; Mawejje, Henry D; Djogbenou, Luc S; Steen, Keith; Rippon, Emily J; Clarkson, Christopher S; Field, Stuart G; Rigden, Daniel J; Donnelly, Martin J

2015-06-01

Functionally constrained genes are ideal insecticide targets because disruption is often fatal, and resistance mutations are typically costly. Synaptic acetylcholinesterase (AChE) is an essential neurotransmission enzyme targeted by insecticides used increasingly in malaria control. In Anopheles and Culex mosquitoes, a glycine-serine substitution at codon 119 of the Ace-1 gene confers both resistance and fitness costs, especially for 119S/S homozygotes. G119S in Anopheles gambiae from Accra (Ghana) is strongly associated with resistance, and, despite expectations of cost, resistant 119S alleles are increasing significantly in frequency. Sequencing of Accra females detected only a single Ace-1 119S haplotype, whereas 119G diversity was high overall but very low at non-synonymous sites, evidence of strong purifying selection driven by functional constraint. Flanking microsatellites showed reduced diversity, elevated linkage disequilibrium and high differentiation of 119S, relative to 119G homozygotes across up to two megabases of the genome. Yet these signals of selection were inconsistent and sometimes weak tens of kilobases from Ace-1. This unexpected finding is attributable to apparently ubiquitous amplification of 119S alleles as part of a large copy number variant (CNV) far exceeding the size of the Ace-1 gene, whereas 119G alleles were unduplicated. Ace-1 CNV was detectable in archived samples collected when the 119S allele was rare in Ghana. Multicopy amplification of resistant alleles has not been observed previously and is likely to underpin the recent increase in 119S frequency. The large CNV compromised localization of the strong selective sweep around Ace-1, emphasizing the need to integrate CNV analysis into genome scans for selection. © 2015 The Authors. Molecular Ecology published by John Wiley & Sons Ltd.

Detection of a lysosomal carboxypeptidase and a lysosomal dipeptidase in highly-purified dipeptidyl aminopeptidase I /cathepsin C/ and the elimination of their activities from preparations used to sequence peptides.

NASA Technical Reports Server (NTRS)

Mcdonald, J. K.; Zeitman, B. B.; Ellis, S.

1972-01-01

Description of the properties of a carboxypeptidase, termed 'catheptic carboxypeptidase C,' and a dipeptidase, termed 'Ser-Met dipeptidase.' Both were found in highly purified DAP I from either beef spleen or rat liver. Methods are described for the removal or selective inactivation of these contaminating enzymes.
Mitigating Mitochondrial Genome Erosion Without Recombination.

PubMed

Radzvilavicius, Arunas L; Kokko, Hanna; Christie, Joshua R

2017-11-01

Mitochondria are ATP-producing organelles of bacterial ancestry that played a key role in the origin and early evolution of complex eukaryotic cells. Most modern eukaryotes transmit mitochondrial genes uniparentally, often without recombination among genetically divergent organelles. While this asymmetric inheritance maintains the efficacy of purifying selection at the level of the cell, the absence of recombination could also make the genome susceptible to Muller's ratchet. How mitochondria escape this irreversible defect accumulation is a fundamental unsolved question. Occasional paternal leakage could in principle promote recombination, but it would also compromise the purifying selection benefits of uniparental inheritance. We assess this tradeoff using a stochastic population-genetic model. In the absence of recombination, uniparental inheritance of freely-segregating genomes mitigates mutational erosion, while paternal leakage exacerbates the ratchet effect. Mitochondrial fusion-fission cycles ensure independent genome segregation, improving purifying selection. Paternal leakage provides opportunity for recombination to slow down the mutation accumulation, but always at a cost of increased steady-state mutation load. Our findings indicate that random segregation of mitochondrial genomes under uniparental inheritance can effectively combat the mutational meltdown, and that homologous recombination under paternal leakage might not be needed. Copyright © 2017 by the Genetics Society of America.
Purification and characterization of an endoglucanase from the marine rotifer, Brachionus plicatilis.

PubMed

Chun, C Z; Hur, S B; Kim, Y T

1997-10-01

The marine rotifer, Brachionus plicatilis, is able to digest Chlorella efficiently, suggesting that the rotifer contains a powerful cellulolytic enzyme system. A multi-component cellulolytic complex, including endoglucanase (CM-cellulase), cellobiohydrolase and beta-glucosidase, was found in Brachionus plicatilis. Endoglucanase (endo-beta-1,4 glucanase) was purified to homogeneity from rotifer homogenates using a sequential chromatographic method. The purified enzyme exhibits a strong hydrolytic activity with carboxymethyl(CM)-cellulose. The optimum temperature and pH for the endoglucanase activity were 37 degrees C and 7.0, respectively. 80% of the CM-cellulase activity was retained in salt mixture that ranged from 150 to 500 mM NaCl equivalent. The purified protein was isolated with a molecular weight of approximately 62 kDa estimated by SDS-polyacrylamide gel electrophoresis.
Vitellogenin from the Silkworm, Bombyx mori: An Effective Anti-Bacterial Agent

PubMed Central

Kumar, Manish; Prasad, Tulika; Kannan, Mani; König, Simone

2013-01-01

Silkworm, Bombyx mori, vitellogenin (Vg) was isolated from perivisceral fat body of day 3 of pupa. Both Vg subunits were co-purified as verified by mass spectrometry and immunoblot. Purified Vg responded to specific tests for major posttranslational modifications on native gels indicating its nature as lipo-glyco-phosphoprotein. The Vg fraction had strong antibacterial activity against Gram negative bacterium Escherichia coli and Gram positive bacterium Bacillus subtilis. Microscopic images showed binding of Vg to bacterial cells and their destruction. When infected silkworm larvae were treated with purified Vg they survived the full life cycle in contrast to untreated animals. This result showed that Vg has the ability to inhibit the proliferation of bacteria in the silkworm fluid system without disturbing the regular metabolism of the host. PMID:24058454
Vitellogenin from the silkworm, Bombyx mori: an effective anti-bacterial agent.

PubMed

Singh, Nitin Kumar; Pakkianathan, Britto Cathrin; Kumar, Manish; Prasad, Tulika; Kannan, Mani; König, Simone; Krishnan, Muthukalingan

2013-01-01

Silkworm, Bombyx mori, vitellogenin (Vg) was isolated from perivisceral fat body of day 3 of pupa. Both Vg subunits were co-purified as verified by mass spectrometry and immunoblot. Purified Vg responded to specific tests for major posttranslational modifications on native gels indicating its nature as lipo-glyco-phosphoprotein. The Vg fraction had strong antibacterial activity against Gram negative bacterium Escherichia coli and Gram positive bacterium Bacillus subtilis. Microscopic images showed binding of Vg to bacterial cells and their destruction. When infected silkworm larvae were treated with purified Vg they survived the full life cycle in contrast to untreated animals. This result showed that Vg has the ability to inhibit the proliferation of bacteria in the silkworm fluid system without disturbing the regular metabolism of the host.
Involvement of an Extracellular Protease in Algicidal Activity of the Marine Bacterium Pseudoalteromonas sp. Strain A28

PubMed Central

Lee, Sun-og; Kato, Junichi; Takiguchi, Noboru; Kuroda, Akio; Ikeda, Tsukasa; Mitsutani, Atsushi; Ohtake, Hisao

2000-01-01

The marine bacterium Pseudoalteromonas sp. strain A28 was able to kill the diatom Skeletonema costatum strain NIES-324. The culture supernatant of strain A28 showed potent algicidal activity when it was applied to a paper disk placed on a lawn of S. costatum NIES-324. The condensed supernatant, which was prepared by subjecting the A28 culture supernatant to ultrafiltration with a 10,000-Mw-cutoff membrane, showed algicidal activity, suggesting that strain A28 produced extracellular substances capable of killing S. costatum cells. The condensed supernatant was then found to have protease and DNase activities. Two Pseudoalteromonas mutants lacking algicidal activity, designated NH1 and NH2, were selected after N-methyl-N′-nitrosoguanidine mutagenesis. The culture supernatants of NH1 and NH2 showed less than 15% of the protease activity detected with the parental strain, A28. The protease was purified to homogeneity from A28 culture supernatants by using ion-exchange chromatography followed by preparative gel electrophoresis. Paper-disk assays revealed that the purified protease had potent algicidal activity. The purified protease had a molecular mass for 50 kDa, and the N-terminal amino acid sequence was determined to be Ala-Thr-Pro-Asn-Asp-Pro. The optimum pH and temperature of the protease were found to be 8.8 and 30°C, respectively, by using succinyl-Ala-Ala-Pro-Phe-p-nitroanilide as a substrate. The protease activity was strongly inhibited by phenylmethylsulfonyl fluoride, diisopropyl fluorophosphate, antipain, chymostatin, and leupeptin. No significant inhibition was detected with EDTA, EGTA, phenanthroline or tetraethylenepentamine. These results suggest that Pseudoalteromonas sp. strain A28 produced an extracellular serine protease which was responsible for the algicidal activity of this marine bacterium. PMID:11010878
Population genomics of the inbred Scandinavian wolf.

PubMed

Hagenblad, Jenny; Olsson, Maria; Parker, Heidi G; Ostrander, Elaine A; Ellegren, Hans

2009-04-01

The Scandinavian wolf population represents one of the genetically most well-characterized examples of a severely bottlenecked natural population (with only two founders), and of how the addition of new genetic material (one immigrant) can at least temporarily provide a 'genetic rescue'. However, inbreeding depression has been observed in this population and in the absence of additional immigrants, its long-term viability is questioned. To study the effects of inbreeding and selection on genomic diversity, we performed a genomic scan with approximately 250 microsatellite markers distributed across all autosomes and the X chromosome. We found linkage disequilibrium (LD) that extended up to distances of 50 Mb, exceeding that of most outbreeding species studied thus far. LD was particularly pronounced on the X chromosome. Overall levels of observed genomic heterozygosity did not deviate significantly from simulations based on known population history, giving no support for a general selection for heterozygotes. However, we found evidence supporting balancing selection at a number of loci and also evidence suggesting directional selection at other loci. For markers on chromosome 23, the signal of selection was particularly strong, indicating that purifying selection against deleterious alleles may have occurred even in this very small population. These data suggest that population genomics allows the exploration of the effects of neutral and non-neutral evolution on a finer scale than what has previously been possible.
Purification and characterization of myrosinase from horseradish (Armoracia rusticana) roots.

PubMed

Li, Xian; Kushad, Mosbah M

2005-06-01

Myrosinase (beta-thioglucoside glucohydrolase; EC 3.2.3.147) from horseradish (Armoracia rusticana) roots was purified to homogeneity by ammonium sulfate fractionation, Q-sepharose, and concanavalin A sepharose affinity chromatography. The purified protein migrated as a single band with a mass of about 65 kDa on SDS-polyacrylamide gel electrophoresis. Using LC-MS/MS, this band was identified as myrosinase. Western blot analysis, using the anti-myrosinase monoclonal antibody 3D7, showed a single band of about 65 kDa for horseradish crude extract and for the purified myrosinase. The native molecular mass of the purified myrosinase was estimated, using gel filtration, to be about 130 kDa. Based on these data, it appeared that myrosinase from horseradish root consists of two subunits of similar molecular mass of about 65 kDa. The enzyme exhibited high activity at broad pH (pH 5.0-8.0) and temperature (37 and 45 degrees C). The purified enzyme remained stable at 4 degrees C for more than 1 year. Using sinigrin as a substrate, the Km and Vmax values for the purified enzyme were estimated to be 0.128 mM and 0.624 micromol min(-1), respectively. The enzyme was strongly activated by 0.5 mM ascorbic acid and was able to breakdown intact glucosinolates in a crude extract of broccoli.
Nucleotide substitutions in dengue virus serotypes from Asian and American countries: insights into intracodon recombination and purifying selection

PubMed Central

2013-01-01

Background Dengue virus (DENV) infection represents a significant public health problem in many subtropical and tropical countries. Although genetically closely related, the four serotypes of DENV differ in antigenicity for which cross protection among serotypes is limited. It is also believed that both multi-serotype infection as well as the evolution of viral antigenicity may have confounding effects in increased dengue epidemics. Numerous studies have been performed that investigated genetic diversity of DENV, but the precise mechanism(s) of dengue virus evolution are not well understood. Results We investigated genome-wide genetic diversity and nucleotide substitution patterns in the four serotypes among samples collected from different countries in Asia and Central and South America and sequenced as part of the Genome Sequencing Center for Infectious Diseases at the Broad Institute. We applied bioinformatics, statistical and coalescent simulation methods to investigate diversity of codon sequences of DENV samples representing the four serotypes. We show that fixation of nucleotide substitutions is more prominent among the inter-continental isolates (Asian and American) of serotypes 1, 2 and 3 compared to serotype 4 isolates (South and Central America) and are distributed in a non-random manner among the genes encoded by the virus. Nearly one third of the negatively selected sites are associated with fixed mutation sites within serotypes. Our results further show that of all the sites showing evidence of recombination, the majority (~84%) correspond to sites under purifying selection in the four serotypes. The analysis further shows that genetic recombination occurs within specific codons, albeit with low frequency (< 5% of all recombination sites) throughout the DENV genome of the four serotypes and reveals significant enrichment (p < 0.05) among sites under purifying selection in the virus. Conclusion The study provides the first evidence for intracodon recombination in DENV and suggests that within codons, genetic recombination has a significant role in maintaining extensive purifying selection of DENV in natural populations. Our study also suggests that fixation of beneficial mutations may lead to virus evolution via translational selection of specific sites in the DENV genome. PMID:23410119
Evolution of High Mobility Group Nucleosome-Binding Proteins and Its Implications for Vertebrate Chromatin Specialization

PubMed Central

González-Romero, Rodrigo; Eirín-López, José M.; Ausió, Juan

2015-01-01

High mobility group (HMG)-N proteins are a family of small nonhistone proteins that bind to nucleosomes (N). Despite the amount of information available on their structure and function, there is an almost complete lack of information on the molecular evolutionary mechanisms leading to their exclusive differentiation. In the present work, we provide evidence suggesting that HMGN lineages constitute independent monophyletic groups derived from a common ancestor prior to the diversification of vertebrates. Based on observations of the functional diversification across vertebrate HMGN proteins and on the extensive silent nucleotide divergence, our results suggest that the long-term evolution of HMGNs occurs under strong purifying selection, resulting from the lineage-specific functional constraints of their different protein domains. Selection analyses on independent lineages suggest that their functional specialization was mediated by bursts of adaptive selection at specific evolutionary times, in a small subset of codons with functional relevance—most notably in HMGN1, and in the rapidly evolving HMGN5. This work provides useful information to our understanding of the specialization imparted on chromatin metabolism by HMGNs, especially on the evolutionary mechanisms underlying their functional differentiation in vertebrates. PMID:25281808
Polymorphism at the defensin gene in the Anopheles gambiae complex: testing different selection hypotheses

PubMed Central

Simard, Frédéric; Licht, Monica; Besansky, Nora J.; Lehmann, Tovi

2007-01-01

Genetic variation in defensin, a gene encoding a major effector molecule of insects immune response was analyzed within and between populations of three members of the Anopheles gambiae complex. The species selected included the two anthropophilic species, An. gambiae and An. arabiensis and the most zoophilic species of the complex, An. quadriannulatus. The first species was represented by four populations spanning its extreme genetic and geographical ranges, whereas each of the other two species was represented by a single population. We found (i) reduced overall polymorphism in the mature peptide region and in the total coding region, together with specific reductions in rare and moderately frequent mutations (sites) in the coding region compared with non coding regions, (ii) markedly reduced rate of nonsynonymous diversity compared with synonymous variation in the mature peptide and virtually identical mature peptide across the three species, and (iii) increased divergence between species in the mature peptide together with reduced differentiation between populations of An. gambiae in the same DNA region. These patterns suggest a strong purifying selection on the mature peptide and probably the whole coding region. Because An. quadriannulatus is not exposed to human pathogens, identical mature peptide and similar pattern of polymorphism across species implies that human pathogens played no role as selective agents on this peptide. PMID:17161659
Subcomponent vaccine based on CTA1-DD adjuvant with incorporated UreB class II peptides stimulates protective Helicobacter pylori immunity.

PubMed

Nedrud, John G; Bagheri, Nayer; Schön, Karin; Xin, Wei; Bergroth, Hilda; Eliasson, Dubravka Grdic; Lycke, Nils Y

2013-01-01

A mucosal vaccine against Helicobacter pylori infection could help prevent gastric cancers and peptic ulcers. While previous attempts to develop such a vaccine have largely failed because of the requirement for safe and effective adjuvants or large amounts of well defined antigens, we have taken a unique approach to combining our strong mucosal CTA1-DD adjuvant with selected peptides from urease B (UreB). The protective efficacy of the selected peptides together with cholera toxin (CT) was first confirmed. However, CT is a strong adjuvant that unfortunately is precluded from clinical use because of its toxicity. To circumvent this problem we have developed a derivative of CT, the CTA1-DD adjuvant, that has been found safe in non-human primates and equally effective compared to CT when used intranasally. We genetically fused the selected peptides into the CTA1-DD plasmid and found after intranasal immunizations of Balb/c mice using purified CTA1-DD with 3 copies of an H. pylori urease T cell epitope (CTA1-UreB3T-DD) that significant protection was stimulated against a live challenge infection. Protection was, however, weaker than with the gold standard, bacterial lysate+CT, but considering that we only used a single epitope in nanomolar amounts the results convey optimism. Protection was associated with enhanced Th1 and Th17 immunity, but immunizations in IL-17A-deficient mice revealed that IL-17 may not be essential for protection. Taken together, we have provided evidence for the rational design of an effective mucosal subcomponent vaccine against H. pylori infection based on well selected protective epitopes from relevant antigens incorporated into the CTA1-DD adjuvant platform.
Subcomponent Vaccine Based on CTA1-DD Adjuvant with Incorporated UreB Class II Peptides Stimulates Protective Helicobacter pylori Immunity

PubMed Central

Nedrud, John G.; Bagheri, Nayer; Schön, Karin; Xin, Wei; Bergroth, Hilda; Eliasson, Dubravka Grdic; Lycke, Nils Y.

2013-01-01

A mucosal vaccine against Helicobacter pylori infection could help prevent gastric cancers and peptic ulcers. While previous attempts to develop such a vaccine have largely failed because of the requirement for safe and effective adjuvants or large amounts of well defined antigens, we have taken a unique approach to combining our strong mucosal CTA1-DD adjuvant with selected peptides from urease B (UreB). The protective efficacy of the selected peptides together with cholera toxin (CT) was first confirmed. However, CT is a strong adjuvant that unfortunately is precluded from clinical use because of its toxicity. To circumvent this problem we have developed a derivative of CT, the CTA1-DD adjuvant, that has been found safe in non-human primates and equally effective compared to CT when used intranasally. We genetically fused the selected peptides into the CTA1-DD plasmid and found after intranasal immunizations of Balb/c mice using purified CTA1-DD with 3 copies of an H. pylori urease T cell epitope (CTA1-UreB3T-DD) that significant protection was stimulated against a live challenge infection. Protection was, however, weaker than with the gold standard, bacterial lysate+CT, but considering that we only used a single epitope in nanomolar amounts the results convey optimism. Protection was associated with enhanced Th1 and Th17 immunity, but immunizations in IL-17A-deficient mice revealed that IL-17 may not be essential for protection. Taken together, we have provided evidence for the rational design of an effective mucosal subcomponent vaccine against H. pylori infection based on well selected protective epitopes from relevant antigens incorporated into the CTA1-DD adjuvant platform. PMID:24391754
Positive selection and ancient duplications in the evolution of class B floral homeotic genes of orchids and grasses

PubMed Central

Mondragón-Palomino, Mariana; Hiese, Luisa; Härter, Andrea; Koch, Marcus A; Theißen, Günter

2009-01-01

Background Positive selection is recognized as the prevalence of nonsynonymous over synonymous substitutions in a gene. Models of the functional evolution of duplicated genes consider neofunctionalization as key to the retention of paralogues. For instance, duplicate transcription factors are specifically retained in plant and animal genomes and both positive selection and transcriptional divergence appear to have played a role in their diversification. However, the relative impact of these two factors has not been systematically evaluated. Class B MADS-box genes, comprising DEF-like and GLO-like genes, encode developmental transcription factors essential for establishment of perianth and male organ identity in the flowers of angiosperms. Here, we contrast the role of positive selection and the known divergence in expression patterns of genes encoding class B-like MADS-box transcription factors from monocots, with emphasis on the family Orchidaceae and the order Poales. Although in the monocots these two groups are highly diverse and have a strongly canalized floral morphology, there is no information on the role of positive selection in the evolution of their distinctive flower morphologies. Published research shows that in Poales, class B-like genes are expressed in stamens and in lodicules, the perianth organs whose identity might also be specified by class B-like genes, like the identity of the inner tepals of their lily-like relatives. In orchids, however, the number and pattern of expression of class B-like genes have greatly diverged. Results The DEF-like genes from Orchidaceae form four well-supported, ancient clades of orthologues. In contrast, orchid GLO-like genes form a single clade of ancient orthologues and recent paralogues. DEF-like genes from orchid clade 2 (OMADS3-like genes) are under less stringent purifying selection than the other orchid DEF-like and GLO-like genes. In comparison with orchids, purifying selection was less stringent in DEF-like and GLO-like genes from Poales. Most importantly, positive selection took place before the major organ reduction and losses in the floral axis that eventually yielded the zygomorphic grass floret. Conclusion In DEF-like genes of Poales, positive selection on the region mediating interactions with other proteins or DNA could have triggered the evolution of the regulatory mechanisms behind the development of grass-specific reproductive structures. Orchidaceae show a different trend, where gene duplication and transcriptional divergence appear to have played a major role in the canalization and modularization of perianth development. PMID:19383167
Arrayed antibody library technology for therapeutic biologic discovery.

PubMed

Bentley, Cornelia A; Bazirgan, Omar A; Graziano, James J; Holmes, Evan M; Smider, Vaughn V

2013-03-15

Traditional immunization and display antibody discovery methods rely on competitive selection amongst a pool of antibodies to identify a lead. While this approach has led to many successful therapeutic antibodies, targets have been limited to proteins which are easily purified. In addition, selection driven discovery has produced a narrow range of antibody functionalities focused on high affinity antagonism. We review the current progress in developing arrayed protein libraries for screening-based, rather than selection-based, discovery. These single molecule per microtiter well libraries have been screened in multiplex formats against both purified antigens and directly against targets expressed on the cell surface. This facilitates the discovery of antibodies against therapeutically interesting targets (GPCRs, ion channels, and other multispanning membrane proteins) and epitopes that have been considered poorly accessible to conventional discovery methods. Copyright © 2013. Published by Elsevier Inc.
High mobility high efficiency organic films based on pure organic materials

DOEpatents

Salzman, Rhonda F [Ann Arbor, MI; Forrest, Stephen R [Ann Arbor, MI

2009-01-27

A method of purifying small molecule organic material, performed as a series of operations beginning with a first sample of the organic small molecule material. The first step is to purify the organic small molecule material by thermal gradient sublimation. The second step is to test the purity of at least one sample from the purified organic small molecule material by spectroscopy. The third step is to repeat the first through third steps on the purified small molecule material if the spectroscopic testing reveals any peaks exceeding a threshold percentage of a magnitude of a characteristic peak of a target organic small molecule. The steps are performed at least twice. The threshold percentage is at most 10%. Preferably the threshold percentage is 5% and more preferably 2%. The threshold percentage may be selected based on the spectra of past samples that achieved target performance characteristics in finished devices.
Sequence analyses of the distal-less homeobox gene family in East African cichlid fishes reveal signatures of positive selection.

PubMed

Diepeveen, Eveline T; Kim, Fabienne D; Salzburger, Walter

2013-07-17

Gen(om)e duplication events are hypothesized as key mechanisms underlying the origin of phenotypic diversity and evolutionary innovation. The diverse and species-rich lineage of teleost fishes is a renowned example of this scenario, because of the fish-specific genome duplication. Gene families, generated by this and other gene duplication events, have been previously found to play a role in the evolution and development of innovations in cichlid fishes - a prime model system to study the genetic basis of rapid speciation, adaptation and evolutionary innovation. The distal-less homeobox genes are particularly interesting candidate genes for evolutionary novelties, such as the pharyngeal jaw apparatus and the anal fin egg-spots. Here we study the dlx repertoire in 23 East African cichlid fishes to determine the rate of evolution and the signatures of selection pressure. Four intact dlx clusters were retrieved from cichlid draft genomes. Phylogenetic analyses of these eight dlx loci in ten teleost species, followed by an in-depth analysis of 23 East African cichlid species, show that there is disparity in the rates of evolution of the dlx paralogs. Dlx3a and dlx4b are the fastest evolving dlx genes, while dlx1a and dlx6a evolved more slowly. Subsequent analyses of the nonsynonymous-synonymous substitution rate ratios indicate that dlx3b, dlx4a and dlx5a evolved under purifying selection, while signs of positive selection were found for dlx1a, dlx2a, dlx3a and dlx4b. Our results indicate that the dlx repertoire of teleost fishes and cichlid fishes in particular, is shaped by differential selection pressures and rates of evolution after gene duplication. Although the divergence of the dlx paralogs are putative signs of new or altered functions, comparisons with available expression patterns indicate that the three dlx loci under strong purifying selection, dlx3b, dlx4a and dlx5a, are transcribed at high levels in the cichlids' pharyngeal jaw and anal fin. The dlx paralogs emerge as excellent candidate genes for the development of evolutionary innovations in cichlids, although further functional analyses are necessary to elucidate their respective contribution.
Cross-resistance to purified Bt proteins, Bt corn and Bt cotton in a Cry2Ab2-corn resistant strain of Spodoptera frugiperda.

PubMed

Yang, Fei; Kerns, David L; Head, Graham P; Price, Paula; Huang, Fangneng

2017-12-01

Gene-pyramiding by combining two or more dissimilar Bacillus thuringiensis (Bt) proteins into a crop has been used to delay insect resistance. The durability of gene-pyramiding can be reduced by cross-resistance. Fall armyworm, Spodoptera frugiperda, is a major target pest of the Cry2Ab2 protein used in pyramided Bt corn and cotton. Here, we provide the first experimental evaluation of cross-resistance in S. frugiperda selected with Cry2Ab2 corn to multiple Bt sources including purified Bt proteins, Bt corn and Bt cotton. Concentration - response bioassays showed that resistance ratios for Cry2Ab2-resistant (RR) relative to Cry2Ab2-susceptible (SS) S. frugiperda were -1.4 for Cry1F, 1.2 for Cry1A.105, >26.7 for Cry2Ab2, >10.0 for Cry2Ae and -1.1 for Vip3A. Larvae of Cry2Ab2-heterozygous (RS), SS and RR S. frugiperda were all susceptible to Bt corn and Bt cotton containing Cry1 (Cry1F or Cry1A.105) and/or Vip3A proteins. Pyramided Bt cotton containing Cry1Ac + Cry2Ab2 or Cry1Ab + Cry2Ae were also effective against SS and RS, but not RR. These findings suggest that Cry2Ab2-corn-selected S. frugiperda is not cross-resistant to Cry1F, Cry1A.105 or Vip3A protein, or corn and cotton plants containing these Bt proteins, but it can cause strong cross-resistance to Cry2Ae and Bt crops expressing similar Bt proteins. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.
Lectin purified from Musca domestica pupa up-regulates NO and iNOS production via TLR4/NF-κB signaling pathway in macrophages.

PubMed

Cao, Xiaohong; Zhou, Minghui; Wang, Chunling; Hou, Lihua; Zeng, Bin

2011-04-01

The present study reported that nitric oxide (NO) was up-regulated by the induction of lectin purified from Musca domestica pupa (MPL) in macrophages without cytotoxicity. The mRNA expression and protein secretion of inducible nitric oxide synthase (iNOS) were strongly induced by MPL treatments. Subsequent investigation revealed that the nuclear factor-κB (NF-κB) inhibitory κB (IκB) in endochylema was inhibited and NF-κB translocated into the nucleus after MPL treatment. Meanwhile, the IKKβ was strongly induced and the production of the toll-like receptor 4 (TLR4) was significantly up-regulated. Moreover, MPL increased NO production via inducing the expression of iNOS through the activation of NF-κB, which suggested that MPL probably acted as an activating agent of the NF-κB activation. Copyright © 2010 Elsevier B.V. All rights reserved.
Compression selective solid-state chemistry

NASA Astrophysics Data System (ADS)

Hu, Anguang

Compression selective solid-state chemistry refers to mechanically induced selective reactions of solids under thermomechanical extreme conditions. Advanced quantum solid-state chemistry simulations, based on density functional theory with localized basis functions, were performed to provide a remarkable insight into bonding pathways of high-pressure chemical reactions in all agreement with experiments. These pathways clearly demonstrate reaction mechanisms in unprecedented structural details, showing not only the chemical identity of reactive intermediates but also how atoms move along the reaction coordinate associated with a specific vibrational mode, directed by induced chemical stress occurred during bond breaking and forming. It indicates that chemical bonds in solids can break and form precisely under compression as we wish. This can be realized through strongly coupling of mechanical work to an initiation vibrational mode when all other modes can be suppressed under compression, resulting in ultrafast reactions to take place isothermally in a few femtoseconds. Thermodynamically, such reactions correspond to an entropy minimum process on an isotherm where the compression can force thermal expansion coefficient equal to zero. Combining a significantly brief reaction process with specific mode selectivity, both statistical laws and quantum uncertainty principle can be bypassed to precisely break chemical bonds, establishing fundamental principles of compression selective solid-state chemistry. Naturally this leads to understand the ''alchemy'' to purify, grow, and perfect certain materials such as emerging novel disruptive energetics.

Selection and paucity of phylogenetic signal challenge the utility of alpha-tubulin in reconstruction of evolutionary history of free-living litostomateans (Protista, Ciliophora).

PubMed

Rajter, Ľubomír; Vďačný, Peter

2018-05-12

The class Litostomatea represents a highly diverse but monophyletic group, uniting both free-living and endosymbiotic ciliates. Ribosomal RNA genes and ITS-region sequences helped to recognize and define the main litostomatean lineages, but did not provide enough phylogenetic signal to unambiguously resolve their interrelationships. In this study, we attempted to improve the resolution among main free-living predatory lineages by adding the gene coding for alpha-tubulin. However, our phylogenetic analyses challenged the performance of alpha-tubulin in reconstruction of evolutionary history of free-living litostomateans. We identified several mutually interconnected problems associated with the ciliate alpha-tubulin gene: the paucity of phylogenetic signal, molecular homoplasies and non-neutral evolution. Positive selection may generate molecular homoplasies (parallel evolution), while negative selection may cause a small number of changes and hence little phylogenetic informativness. Both problems were encountered in nucleotide and amino acid alpha-tubulin alignments, indicating an action of various selective pressures. Taking into account the involvement of alpha-tubulin in many essential biological processes, this protein could be so strongly affected by purifying selection that it even might have become an inappropriate molecular marker for reconstruction of phylogenetic relationships. Therefore, a great caution should be paid when tubulin genes are included in phylogenetic and/or phylogenomic analyses. Copyright © 2018 Elsevier Inc. All rights reserved.
Cytotoxic and antiproliferative effects induced by a non terpenoid polar extract of A. indica seeds on 3T6 murine fibroblasts in culture.

PubMed

Di Ilio, Vincenzo; Pasquariello, Nicoletta; van der Esch, Andrew S; Cristofaro, Massimo; Scarsella, Gianfranco; Risuleo, Gianfranco

2006-07-01

Neem oil is a natural product obtained from the seeds of the tree Azadirachta indica. Its composition is very complex and the oil exhibits a number of biological activities. The most studied component is the terpenoid azadirachtin which is used for its insecticidal and putative antimicrobial properties. In this report we investigate the biological activity of partially purified components of the oil obtained from A. indica. We show that the semi-purified fractions have moderate to strong cytotoxicity. However, this is not attributable to azadirachtin but to other active compounds present in the mixture. Each fraction was further purified by appropriate extraction procedures and we observed a differential cytotoxicity in the various sub-fractions. This led us to investigate the mode of cell death. After treatment with the oil fractions we observed positivity to TUNEL staining and extensive internucleosomal DNA degradation both indicating apoptotic death. The anti-proliferative properties of the neem oil-derived compounds were also assayed by evaluation of the nuclear PCNA levels (Proliferating Cell Nuclear Antigen). PCNA is significantly reduced in cells treated with a specific fraction of neem oil. Finally, our results strongly suggest a possible involvement of the mitochondrial pathway in the apoptotic death.
Formate Dehydrogenase from Clostridium acidiurici

PubMed Central

Kearny, James J.; Sagers, Richard D.

1972-01-01

Partial purification of formate dehydrogenase from Clostridium acidiurici has been accomplished, and some properties of the enzyme have been determined. The molecular weight of the protein is at least 200,000 daltons. The enzyme showed marked instability to freezing and thawing and was inhibited strongly by oxygen and by light. Such inhibition was not reversed by incubation in the presence of thiol compounds. Cyanide inhibited the enzyme 90% at 0.1 mm concentrations, but ethylenediaminetetraacetate produced only slight inhibition at concentrations as high as 50 mm. The purified enzyme showed no ferredoxin activity in the Clostridium pasteurianum clastic system during pyruvate oxidation. Crude preparations of the enzyme could be coupled through ferredoxin to the reduction of nicotinamide adenine dinucleotide during formate oxidation, but the purified enzyme could not catalyze the reduction of pyridine nucleotides by formate in the presence of ferredoxin. Formate oxidation with the purified enzyme was readily coupled to benzyl viologen reduction, in which case ferredoxin was not required. An exchange between formate and bicarbonate was catalyzed by both crude and purified preparations of the enzyme, but the net synthesis of formate from CO2 was not accomplished. PMID:4333376
Hemizygosity Enhances Purifying Selection: Lack of Fast-Z Evolution in Two Satyrine Butterflies.

PubMed

Rousselle, Marjolaine; Faivre, Nicolas; Ballenghien, Marion; Galtier, Nicolas; Nabholz, Benoit

2016-10-23

The fixation probability of a recessive beneficial mutation is increased on the X or Z chromosome, relative to autosomes, because recessive alleles carried by X or Z are exposed to selection in the heterogametic sex. This leads to an increased dN/dS ratio on sex chromosomes relative to autosomes, a pattern called the "fast-X" or "fast-Z" effect. Besides positive selection, the strength of genetic drift and the efficacy of purifying selection, which affect the rate of molecular evolution, might differ between sex chromosomes and autosomes. Disentangling the complex effects of these distinct forces requires the genome-wide analysis of polymorphism, divergence and gene expression data in a variety of taxa. Here we study the influence of hemizygosity of the Z chromosome in Maniola jurtina and Pyronia tithonus, two species of butterflies (Lepidoptera, Nymphalidae, Satyrinae). Using transcriptome data, we compare the strength of positive and negative selection between Z and autosomes accounting for sex-specific gene expression. We show that M. jurtina and P. tithonus do not experience a faster, but rather a slightly slower evolutionary rate on the Z than on autosomes. Our analysis failed to detect a significant difference in adaptive evolutionary rate between Z and autosomes, but comparison of male-biased, unbiased and female-biased Z-linked genes revealed an increased efficacy of purifying selection against recessive deleterious mutations in female-biased Z-linked genes. This probably contributes to the lack of fast-Z evolution of satyrines. We suggest that the effect of hemizygosity on the fate of recessive deleterious mutations should be taken into account when interpreting patterns of molecular evolution in sex chromosomes vs. autosomes. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.
Background diet and fat type alters plasma lipoprotein response but not aortic cholesterol accumulation in F1B Golden Syrian hamsters.

PubMed

Dillard, Alice; Matthan, Nirupa R; Spartano, Nicole L; Butkowski, Ann E; Lichtenstein, Alice H

2013-12-01

Dietary modification alters plasma lipoprotein profiles and atherosclerotic lesion progression in humans and some animal models. Variability in response to diet induced atherosclerosis has been reported in hamsters. Assessed was the interaction between background diet composition and dietary fat type on aortic cholesterol accumulation, lipoprotein profiles, hepatic lipids and selected genes. F1B Golden Syrian hamsters (20/group) were fed (12 weeks) semi-purified or non-purified diets containing either 10 % (w/w) coconut oil or safflower oil and 0.15 % (w/w) cholesterol. The non-purified diets relative to semi-purified diets resulted in significantly higher TC (72 % [percent difference] and 38 %, coconut oil and safflower oil, respectively) and nHDL-C (84 and 61 %, coconut oil and safflower oil, respectively), and lower HDL-C (-47 and -45 %, coconut oil and safflower oil, respectively) concentrations. Plasma triacylglycerol concentrations in the hamsters fed the non-purified coconut oil-supplemented diets were three- to fourfold higher than non-purified safflower oil-supplemented, and both semi-purified diets. With the exception of HDL-C, a significant effect of fat type was observed in TC, nHDL-C and triacylglycerol (all P < 0.05) concentrations. Regardless of diet induced differences in lipoprotein profiles, there was no significant effect on aortic cholesterol accumulation. There was an inverse relationship between plasma nHDL-C and triacylglycerol, and hepatic cholesteryl ester content (P < 0.001). Diet induced differences in hepatic gene transcription (LDL receptor, apoB-100, microsomal transfer protein) were not reflected in protein concentrations. Although hamsters fed non-purified and/or saturated fatty acid-supplemented diets had more atherogenic lipoprotein profiles compared to hamsters fed semi-purified and/or polyunsaturated fatty acid-supplemented diets these differences were not reflected in aortic cholesterol accumulation.
Large-Scale Genomic Analysis of Codon Usage in Dengue Virus and Evaluation of Its Phylogenetic Dependence

PubMed Central

Lara-Ramírez, Edgar E.; Salazar, Ma Isabel; López-López, María de Jesús; Salas-Benito, Juan Santiago; Sánchez-Varela, Alejandro

2014-01-01

The increasing number of dengue virus (DENV) genome sequences available allows identifying the contributing factors to DENV evolution. In the present study, the codon usage in serotypes 1–4 (DENV1–4) has been explored for 3047 sequenced genomes using different statistics methods. The correlation analysis of total GC content (GC) with GC content at the three nucleotide positions of codons (GC1, GC2, and GC3) as well as the effective number of codons (ENC, ENCp) versus GC3 plots revealed mutational bias and purifying selection pressures as the major forces influencing the codon usage, but with distinct pressure on specific nucleotide position in the codon. The correspondence analysis (CA) and clustering analysis on relative synonymous codon usage (RSCU) within each serotype showed similar clustering patterns to the phylogenetic analysis of nucleotide sequences for DENV1–4. These clustering patterns are strongly related to the virus geographic origin. The phylogenetic dependence analysis also suggests that stabilizing selection acts on the codon usage bias. Our analysis of a large scale reveals new feature on DENV genomic evolution. PMID:25136631
[Separation and purification of the components in Trachelospermum jasminoides by two dimensional hydrophilic interaction liquid chromatography- reversed-phase liquid chromatography].

PubMed

Jia, Youmei; Cai, Jianfeng; Xin, Huaxia; Feng, Jiatao; Fu, Yanhui; Fu, Qing; Jin, Yu

2017-06-08

A preparative two dimensional hydrophilic interaction liquid chromatography/reversed-phase liquid chromatography (Pre-2D-HILIC/RPLC) method was established to separate and purify the components in Trachelospermum jasminoides . The pigments and strongly polar components were removed from the crude extract after the active carbon decolorization and solid phase extraction processes. A Click XIon column (250 mm×20 mm, 10 μm) was selected as stationary phase and water-acetonitrile as mobile phases in the first dimensional HILIC. Finally, 15 fractions were collected under UV-triggered mode. In the second dimensional RPLC, a C18 column (250 mm×20 mm, 5 μm) was selected and water-acetonitrile was used as mobile phases. As a result, 14 compounds with high purity were obtained, which were further identified by mass spectrometry (MS) and nuclear magnetic resonance (NMR). Finally, 11 lignan compounds and three flavonoid compounds were obtained. The method has a good orthogonality, and can improve the resolution and the peak capacity. It is significant for the separation of complex components from Trachelospermum jasminoides .
Strong antimicrobial activity of xanthohumol and other derivatives from hops (Humulus lupulus L.) on gut anaerobic bacteria.

PubMed

Cermak, Pavel; Olsovska, Jana; Mikyska, Alexandr; Dusek, Martin; Kadleckova, Zuzana; Vanicek, Jiri; Nyc, Otakar; Sigler, Karel; Bostikova, Vanda; Bostik, Pavel

2017-11-01

Anaerobic bacteria, such as Bacteroides fragilis or Clostridium perfringens, are part of indigenous human flora. However, Clostridium difficile represents also an important causative agent of nosocomial infectious antibiotic-associated diarrhoea. Treatment of C. difficile infection is problematic, making it imperative to search for new compounds with antimicrobial properties. Hops (Humulus lupulus L.) contain substances with antibacterial properties. We tested antimicrobial activity of purified hop constituents humulone, lupulone and xanthohumol against anaerobic bacteria. The antimicrobial activity was established against B. fragilis, C. perfringens and C. difficile strains according to standard testing protocols (CLSI, EUCAST), and the minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBC) were calculated. All C. difficile strains were toxigenic and clinically relevant, as they were isolated from patients with diarrhoea. Strongest antimicrobial effects were observed with xanthohumol showing MIC and MBC values of 15-107 μg/mL, which are close to those of conventional antibiotics in the strains of bacteria with increased resistance. Slightly higher MIC and MBC values were obtained with lupulone followed by higher values of humulone. Our study, thus, shows a potential of purified hop compounds, especially xanthohumol, as alternatives for treatment of infections caused by select anaerobic bacteria, namely nosocomial diarrhoea caused by resistant strains. © 2017 APMIS. Published by John Wiley & Sons Ltd.
Selective Biological Responses of Phagocytes and Lungs to Purified Histones.

PubMed

Fattahi, Fatemeh; Grailer, Jamison J; Lu, Hope; Dick, Rachel S; Parlett, Michella; Zetoune, Firas S; Nuñez, Gabriel; Ward, Peter A

2017-01-01

Histones invoke strong proinflammatory responses in many different organs and cells. We assessed biological responses to purified or recombinant histones, using human and murine phagocytes and mouse lungs. H1 had the strongest ability in vitro to induce cell swelling independent of requirements for toll-like receptors (TLRs) 2 or 4. These responses were also associated with lactate dehydrogenase release. H3 and H2B were the strongest inducers of [Ca2+]i elevations in phagocytes. Cytokine and chemokine release from mouse and human phagocytes was predominately a function of H2A and H2B. Double TLR2 and TLR4 knockout (KO) mice had dramatically reduced cytokine release induced in macrophages exposed to individual histones. In contrast, macrophages from single TLR-KO mice showed few inhibitory effects on cytokine production. Using the NLRP3 inflammasome protocol, release of mature IL-1β was predominantly a feature of H1. Acute lung injury following the airway delivery of histones suggested that H1, H2A, and H2B were linked to alveolar leak of albumin and the buildup of polymorphonuclear neutrophils as well as the release of chemokines and cytokines into bronchoalveolar fluids. These results demonstrate distinct biological roles for individual histones in the context of inflammation biology and the requirement of both TLR2 and TLR4. © 2017 S. Karger AG, Basel.
The stabilisation of purified, reconstituted P-glycoprotein by freeze drying with disaccharides.

PubMed

Heikal, Adam; Box, Karl; Rothnie, Alice; Storm, Janet; Callaghan, Richard; Allen, Marcus

2009-02-01

The drug efflux pump P-glycoprotein (P-gp) (ABCB1) confers multidrug resistance, a major cause of failure in the chemotherapy of tumours, exacerbated by a shortage of potent and selective inhibitors. A high throughput assay using purified P-gp to screen and characterise potential inhibitors would greatly accelerate their development. However, long-term stability of purified reconstituted ABCB1 can only be reliably achieved with storage at -80 degrees C. For example, at 20 degrees C, the activity of ABCB1 was abrogated with a half-life of <1 day. The aim of this investigation was to stabilise purified, reconstituted ABCB1 to enable storage at higher temperatures and thereby enable design of a high throughput assay system. The ABCB1 purification procedure was optimised to allow successful freeze drying by substitution of glycerol with the disaccharides trehalose or maltose. Addition of disaccharides resulted in ATPase activity being retained immediately following lyophilisation with no significant difference between the two disaccharides. However, during storage trehalose preserved ATPase activity for several months regardless of the temperature (e.g. 60% retention at 150 days), whereas ATPase activity in maltose purified P-gp was affected by both storage time and temperature. The data provide an effective mechanism for the production of resilient purified, reconstituted ABCB1.
Purified Dendritic Cell-Tumor Fusion Hybrids Supplemented with Non-Adherent Dendritic Cells Fraction Are Superior Activators of Antitumor Immunity

PubMed Central

Wang, Yucai; Liu, Yunyan; Zheng, Lianhe

2014-01-01

Background Strong evidence supports the DC-tumor fusion hybrid vaccination strategy, but the best fusion product components to use remains controversial. Fusion products contain DC-tumor fusion hybrids, unfused DCs and unfused tumor cells. Various fractions have been used in previous studies, including purified hybrids, the adherent cell fraction or the whole fusion mixture. The extent to which the hybrids themselves or other components are responsible for antitumor immunity or which components should be used to maximize the antitumor immunity remains unknown. Methods Patient-derived breast tumor cells and DCs were electro-fused and purified. The antitumor immune responses induced by the purified hybrids and the other components were compared. Results Except for DC-tumor hybrids, the non-adherent cell fraction containing mainly unfused DCs also contributed a lot in antitumor immunity. Purified hybrids supplemented with the non-adherent cell population elicited the most powerful antitumor immune response. After irradiation and electro-fusion, tumor cells underwent necrosis, and the unfused DCs phagocytosed the necrotic tumor cells or tumor debris, which resulted in significant DC maturation. This may be the immunogenicity mechanism of the non-adherent unfused DCs fraction. Conclusions The non-adherent cell fraction (containing mainly unfused DCs) from total DC/tumor fusion products had enhanced immunogenicity that resulted from apoptotic/necrotic tumor cell phagocytosis and increased DC maturation. Purified fusion hybrids supplemented with the non-adherent cell population enhanced the antitumor immune responses, avoiding unnecessary use of the tumor cell fraction, which has many drawbacks. Purified hybrids supplemented with the non-adherent cell fraction may represent a better approach to the DC-tumor fusion hybrid vaccination strategy. PMID:24466232
Biocontrol activity of surfactin A purified from Bacillus NH-100 and NH-217 against rice bakanae disease.

PubMed

Sarwar, Ambrin; Hassan, Muhammad Nadeem; Imran, Muhammad; Iqbal, Mazhar; Majeed, Saima; Brader, Günter; Sessitsch, Angela; Hafeez, Fauzia Yusuf

2018-04-01

The potential of the Bacillus genus to antagonize phytopathogens is associated with the production of cyclic lipopeptides. Depending upon the type of lipopeptide, they may serve as biocontrol agents that are eco-friendly alternatives to chemical fertilizers. This study evaluates the biocontrol activity of surfactin-producing Bacillus (SPB) strains NH-100 and NH-217 and purified surfactin A from these strains against rice bakanae disease. Biologically active surfactin fractions were purified by HPLC, and surfactin A variants with chain lengths from C12 to C16 were confirmed by LCMS-ESI. In hemolytic assays, a positive correlation between surfactin A production and halo zone formation was observed. The purified surfactin A had strong antifungal activity against Fusarium oxysporum, F. moniliforme, F. solani, Trichoderma atroviride and T. reesei. Maximum fungal growth suppression (84%) was recorded at 2000 ppm against F. moniliforme. Surfactin A retained antifungal activity at different pH levels (5-9) and temperatures (20, 50 and 121 °C). Hydroponic and pot experiments were conducted to determine the biocontrol activity of SPB strains and the purified surfactin A from these strains on Super Basmati rice. Surfactin production in the rice rhizosphere was detected by LCMS-ESI at early growth stages in hydroponics experiments inoculated with SPB strains. However, the maximum yield was observed with a consortium of SPB strains (T4) and purified surfactin A (T5) treatments in the pot experiment. The outcomes of the present study revealed that surfactin A significantly reduced rice bakanae disease by up to 80%. These findings suggest that purified surfactin A could be an effective biocontrol agent against bakanae disease in rice and should be incorporated into strategies for disease management. Copyright © 2018 Elsevier GmbH. All rights reserved.
Development of a Novel Method for the Purification and Culture of Rodent Astrocytes

PubMed Central

Foo, Lynette C.; Allen, Nicola J.; Bushong, Eric A.; Ventura, P. Britten; Chung, Won-Suk; Zhou, Lu; Cahoy, John D.; Daneman, Richard; Zong, Hui; Ellisman, Mark H.; Barres, Ben A.

2011-01-01

Summary The inability to purify and culture astrocytes has long hindered studies of their function. Whereas astrocyte progenitor cells can be cultured from neonatal brain, culture of mature astrocytes from postnatal brain has not been possible. Here we report a new method to prospectively purify astrocytes by immunopanning. These astrocytes undergo apoptosis in culture, but vascular cells and HBEGF promote their survival in serum-free culture. We found that some developing astrocytes normally undergo apoptosis in vivo and that the vast majority of astrocytes contact blood vessels, suggesting that astrocytes are matched to blood vessels by competing for vascular-derived trophic factors such as HBEGF. Compared to traditional astrocyte cultures, the gene profiles of the cultured purified postnatal astrocytes much more closely resemble those of in vivo astrocytes. Although these astrocytes strongly promote synapse formation and function, they do not secrete glutamate in response to stimulation. PMID:21903074
Characterization, gene cloning, and sequencing of a fungal phytase, PhyA, from Penicillium oxalicum PJ3.

PubMed

Lee, Seung Ho; Cho, Jaiesoon; Bok, Jinduck; Kang, Seungha; Choi, Yunjaie; Lee, Peter C W

2015-01-01

A phytase from Penicillium oxalicum PJ3, PhyA, was purified near to homogeneity with 427-fold increase in specific phytase activity by ammonium sulfate precipitation, gel filtration, and ion-exchange chromatographies. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and zymogram analysis of the purified enzyme indicated an estimated molecular mass of 65 kD. The optimal pH and temperature of the purified enzyme were pH 4.5 and 55°C, respectively. The enzyme activity was strongly inhibited by Ca(2+), Cu(2+), Zn(2+), and phenylmethylsulfonyl fluoride (PMSF). The Km value for sodium phytate was 0.545 mM with a Vmax of 600 U/mg of protein. The phyA gene was cloned, and it contains an open reading frame of 1,383 with a single intron (118 bp), and encodes a protein of 461 amino acids.
Purification and Characterization of a Fibrinolytic Enzyme from Bacillus pumilus 2.g Isolated from Gembus, an Indonesian Fermented Food

PubMed Central

Afifah, Diana Nur; Sulchan, Muhammad; Syah, Dahrul; Yanti; Suhartono, Maggy Thenawidjaja; Kim, Jeong Hwan

2014-01-01

Bacillus pumilus 2.g isolated from gembus, an Indonesian fermented soybean cake, secretes several proteases that have strong fibrinolytic activities. A fibrinolytic enzyme with an apparent molecular weight of 20 kDa was purified from the culture supernatant of B. pumilus 2.g by sequential application of ammonium sulfate precipitation, ion-exchange chromatography, and hydrophobic chromatography. The partially purified enzyme was stable between pH 5 and pH 9 and temperature of less than 60°C. Fibrinolytic activity was increased by 5 mM MgCl2 and 5 mM CaCl2 but inhibited by 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM sodium dodecyl sulfate (SDS), and 1 mM ethylenediaminetetraacetic acid (EDTA). The partially purified enzyme quickly degraded the α and β chains of fibrinogen but was unable to degrade the γ chain. PMID:25320719
In vitro and in vivo antioxidant activities of polysaccharide purified from aloe vera (Aloe barbadensis) gel.

PubMed

Kang, Min-Cheol; Kim, Seo Young; Kim, Yoon Taek; Kim, Eun-A; Lee, Seung-Hong; Ko, Seok-Chun; Wijesinghe, W A J P; Samarakoon, Kalpa W; Kim, Young-Sun; Cho, Jin Hun; Jang, Hyeang-Su; Jeon, You-Jin

2014-01-01

The in vitro and in vivo antioxidant potentials of a polysaccharide isolated from aloe vera gel were investigated. Enzymatic extracts were prepared from aloe vera gel by using ten digestive enzymes including five carbohydrases and five proteases. Among them, the highest yield was obtained with the Viscozyme extract and the same extract showed the best radical scavenging activity. An active polysaccharide was purified from the Viscozyme extract using ethanol-added separation and anion exchange chromatography. Purified aloe vera polysaccharide (APS) strongly scavenged radicals including DPPH, hydroxyl and alkyl radicals. In addition, APS showed a protective effect against AAPH-induced oxidative stress and cell death in Vero cells as well as in the in vivo zebrafish model. In this study, it is proved that both the in vitro and in vivo antioxidant potentials of APS could be further utilized in relevant industrial applications. Copyright © 2013 Elsevier Ltd. All rights reserved.
Contrasting mode of evolution at a coat color locus in wild and domestic pigs.

PubMed

Fang, Meiying; Larson, Greger; Ribeiro, Helena Soares; Li, Ning; Andersson, Leif

2009-01-01

Despite having only begun approximately 10,000 years ago, the process of domestication has resulted in a degree of phenotypic variation within individual species normally associated with much deeper evolutionary time scales. Though many variable traits found in domestic animals are the result of relatively recent human-mediated selection, uncertainty remains as to whether the modern ubiquity of long-standing variable traits such as coat color results from selection or drift, and whether the underlying alleles were present in the wild ancestor or appeared after domestication began. Here, through an investigation of sequence diversity at the porcine melanocortin receptor 1 (MC1R) locus, we provide evidence that wild and domestic pig (Sus scrofa) haplotypes from China and Europe are the result of strikingly different selection pressures, and that coat color variation is the result of intentional selection for alleles that appeared after the advent of domestication. Asian and European wild boar (evolutionarily distinct subspecies) differed only by synonymous substitutions, demonstrating that camouflage coat color is maintained by purifying selection. In domestic pigs, however, each of nine unique mutations altered the amino acid sequence thus generating coat color diversity. Most domestic MC1R alleles differed by more than one mutation from the wild-type, implying a long history of strong positive selection for coat color variants, during which time humans have cherry-picked rare mutations that would be quickly eliminated in wild contexts. This pattern demonstrates that coat color phenotypes result from direct human selection and not via a simple relaxation of natural selective pressures.
DNA adducts of ethylene dibromide: Aspects of formation and mutagenicity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cmarik, J.L.

1,2-Dibromoethane (ethylene dibromide, EDB), a potential human carcinogen, undergoes bioactivation by the pathway of glutathione (GSH) conjugation, which generates a reactive intermediate capable of alkylating DNA. The major DNA adduct formed is S-[2-(N[sup 7]-guanyl)ethyl]GSH. This dissertation examined the bioactivation of EDB and the formation of DNA adducts. The selectivity of purified rat and human GSH S-transferases for EDB was examined in vitro. An assay was developed to measure the formation of S,S[prime]-ethylene-bis(GSH). The [alpha] class of the GSH S-transferases was responsible for the majority of EDB-GSH conjugation with both the rat and human enzymes. Human tissue samples for a victimmore » of EDB poisoning were analyzed for S-[2-(N[sup 7]-guanyl)ethyl]GSH utilizing electrochemical detection. No adducts were detected in samples of brain, heart, or kidney. The pattern of alkylation of guanines in fragments of plasmid pBR322 DNA by S-(2-chloroethyl)GSH and related compounds was determined. Alkylation varied approximately ten-fold in intensity and was strongest in runs of guanines. Few differences were observed in the alkylation patterns generated by the different compounds tested. The spectrum of mutations caused by S-(2-chloroethyl)GSH was determined using an M13 bacteriophage forward mutation assay. The majority of mutations (70%) were G:C to A:T transitions. Participation of the N[sup 7]-guanyl adduct in the mutagenic process is strongly implicated. The sequence selectivity of alkylation in the region of M13 sequenced in the mutation assay was determined. Comparison of the sequence selectivity with the mutation spectrum revealed no obligate relationship between the extent of adduct formation and the number of mutations which resulted at different sites. Sequence context appears to exert a strong influence on the processing of lesions. These studies strongly implicate S-[2-(N[sup 7]-guanyl)-ethyl]GSH as a mutagenic lesion formed by EDB.« less
Population genetics of mouse lemur vomeronasal receptors: current versus past selection and demographic inference.

PubMed

Hohenbrink, Philipp; Mundy, Nicholas I; Radespiel, Ute

2017-01-21

A major effort is underway to use population genetic approaches to identify loci involved in adaptation. One issue that has so far received limited attention is whether loci that show a phylogenetic signal of positive selection in the past also show evidence of ongoing positive selection at the population level. We address this issue using vomeronasal receptors (VRs), a diverse gene family in mammals involved in intraspecific communication and predator detection. In mouse lemurs, we previously demonstrated that both subfamilies of VRs (V1Rs and V2Rs) show a strong signal of directional selection in interspecific analyses. We predicted that ongoing sexual selection and/or co-evolution with predators may lead to current directional or balancing selection on VRs. Here, we re-sequence 17 VRs and perform a suite of selection and demographic analyses in sympatric populations of two species of mouse lemurs (Microcebus murinus and M. ravelobensis) in northwestern Madagascar. M. ravelobensis had consistently higher genetic diversity at VRs than M. murinus. In general, we find little evidence for positive selection, with most loci evolving under purifying selection and one locus even showing evidence of functional loss in M. ravelobensis. However, a few loci in M. ravelobensis show potential evidence of positive selection. Using mismatch distributions and expansion models, we infer a more recent colonisation of the habitat by M. murinus than by M. ravelobensis, which most likely speciated in this region earlier on. These findings suggest that the analysis of VR variation is useful in inferring demographic and phylogeographic history of mouse lemurs. In conclusion, this study reveals a substantial heterogeneity over time in selection on VR loci, suggesting that VR evolution is episodic.
Microevolutionary dynamics of a macroevolutionary key innovation in a Lepidopteran herbivore

PubMed Central

2010-01-01

Background A molecular population genetics understanding is central to the study of ecological and evolutionary functional genomics. Population genetics identifies genetic variation and its distribution within and among populations, it reveals the demographic history of the populations studied, and can provide indirect insights into historical selection dynamics. Here we use this approach to examine the demographic and selective dynamics acting of a candidate gene involved in plant-insect interactions. Previous work documents the macroevolutionary and historical ecological importance of the nitrile-specifier protein (Nsp), which facilitated the host shift of Pieridae butterflies onto Brassicales host plants ~80 Myr ago. Results Here we assess the microevolutionary dynamics of the Nsp gene by studying the within and among-population variation at Nsp and reference genes in the butterfly Pieris rapae (Small Cabbage White). Nsp exhibits unexpectedly high amounts of amino acid polymorphism, unequally distributed across the gene. The vast majority of genetic variation exists within populations, with little to no genetic differentiation among four populations on two continents. A comparison of synonymous and nonsynonymous substitutions in 70 randomly chosen genes among P. rapae and its close relative Pieris brassicae (Large Cabbage White) finds Nsp to have a significantly relaxed functional constraint compared to housekeeping genes. We find strong evidence for a recent population expansion and no role for strong purifying or directional selection upon the Nsp gene. Conclusions The microevolutionary dynamics of the Nsp gene in P. rapae are dominated by recent population expansion and variation in functional constraint across the repeated domains of the Nsp gene. While the high amounts of amino acid diversity suggest there may be significant functional differences among allelic variants segregating within populations, indirect tests of selection could not conclusively identify a signature of historical selection. The importance of using this information for planning future studies of potential performance and fitness consequences of the observed variation is discussed. PMID:20181249

Electrochemical ion separation in molten salts

DOEpatents

Spoerke, Erik David; Ihlefeld, Jon; Waldrip, Karen; Wheeler, Jill S.; Brown-Shaklee, Harlan James; Small, Leo J.; Wheeler, David R.

2017-12-19

A purification method that uses ion-selective ceramics to electrochemically filter waste products from a molten salt. The electrochemical method uses ion-conducting ceramics that are selective for the molten salt cations desired in the final purified melt, and selective against any contaminant ions. The method can be integrated into a slightly modified version of the electrochemical framework currently used in pyroprocessing of nuclear wastes.
Mutation Accumulation, Soft Selection and the Middle-Class Neighborhood

PubMed Central

Moorad, Jacob A.; Hall, David W.

2009-01-01

The “middle-class neighborhood” is a breeding design intended to allow new mutations to accumulate by lessening the effects of purifying selection through the elimination of among-line fitness variation. We show that this design effectively applies soft selection to the experimental population, potentially causing biased estimates of mutational effects if social effects contribute to fitness. PMID:19448272
Phylogeny and evolution of Newcastle disease virus genotypes isolated in Asia during 2008-2011.

PubMed

Ebrahimi, Mohammad Majid; Shahsavandi, Shahla; Moazenijula, Gholamreza; Shamsara, Mahdi

2012-08-01

The full-length fusion (F) genes of 51 Newcastle disease (ND) strains isolated from chickens in Asia during the period 2008-2011 were genetically analyzed. Phylogenetic analysis showed that genotype VII of NDV still predominant in the domestic poultry of Asia. The sub-genotype VIIb circulated in the Iran and Indian sub-continent countries, whereas VIId sub-genotype existed in Far East countries. The non-synonymous to synonymous substitutions ratio was calculated 0.27 for VIId sub-genotype and 0.51 for VIIb sub-genotype indicates purifying/stabilizing selection which resulted in a low evolution rate in F gene of VIIb sub-genotype. There is evidence of localized positive selection when comparing these sub-genotypes protein sequences. Five codons in F gene of ND viruses had a posterior probability >90% using the Bayesian method, indicating these sites were under positive selection. To identify sites under positive selection; amino acid substitution classified depends on their radicalism and neutrality. The results indicate that although most positions were under purifying selection and can be eliminated, a few positions located in sub-genotype specific regions were subject to positive selection.
New Instrumentation for Phase Partitioning

NASA Technical Reports Server (NTRS)

Harris, J. M.

1985-01-01

Cells and molecules can be purified by partitioning between the two immiscible liquid phases formed by aqueous solutions of poly/ethylene glycol and dextran. Such purification can be more selective, higher yielding, and less destructive to sensitive biological materials than other available techniques. Earth's gravitational field is a hindering factor as it causes sedimentation of particles to be purified and shear-induced particle randomization. The present proposal is directed toward developing new instrumentation for performing phase partitioning both on Earth and in microgravity.
Alumina plate containing photosystem I reaction center complex oriented inside plate-penetrating silica nanopores.

PubMed

Kamidaki, Chihiro; Kondo, Toru; Noji, Tomoyasu; Itoh, Tetsuji; Yamaguchi, Akira; Itoh, Shigeru

2013-08-22

The photosynthetic photosystem I reaction center complex (PSI-RC), which has a molecular diameter of 21 nm with 100 pigments, was incorporated into silica nanopores with a 100-nm diameter that penetrates an alumina plate of 60-μm thickness to make up an inorganic-biological hybrid photocell. PSI-RCs, purified from a thermophilic cyanobacterium, were stable inside the nanopores and rapidly photoreduced a mediator dye methyl viologen. The reduced dye was more stable inside nanopores suggesting the decrease of dissolved oxygen. The analysis by a cryogenic electron spin paramagnetic resonance indicated the oriented arrangement of RCs inside the 100-nm nanopores, with their surface parallel to the silica wall and perpendicular to the plane of the alumina plate. PSI RC complex in the semicrystalline orientation inside silica nanopores can be a new type of light energy conversion unit to supply strong reducing power selectively to other molecules inside or outside nanopores.
Purification of Torpedo californica post-synaptic membranes and fractionation of their constituent proteins.

PubMed Central

Elliott, J; Blanchard, S G; Wu, W; Miller, J; Strader, C D; Hartig, P; Moore, H P; Racs, J; Raftery, M A

1980-01-01

A rapid methof for preparation of membrane fractions highly enriched in nicotinic acetylcholine receptor from Torpedo californica electroplax is described. The major step in this purification involves sucrose-density-gradient centrifugation in a reorienting rotor. Further purification of these membranes can be achieved by selective extraction of proteins by use of alkaline pH or by treatment with solutions of lithium di-idosalicylate. The alkali-treated membranes retain functional characteristics of the untreated membranes and in addition contain essentially only the four polypeptides (mol.wts. 40000, 50000, 60000 and 65000) characteristic of the receptor purified by affinity chromatography. Dissolution of the purified membranes or of the alkali-treated purified membranes in sodium cholate solution followed by sucrose-density-gradient centrifugation in the same detergent solution yields solubilized receptor preparations comparable with the most highly purified protein obtained by affinity-chromatographic procedures. Images Fig. 1. Fig. 2. Fig. 3. Fig. 5. Fig. 7. PLATE 1 PMID:7387629
Effect of saposins on acid sphingomyelinase.

PubMed Central

Tayama, M; Soeda, S; Kishimoto, Y; Martin, B M; Callahan, J W; Hiraiwa, M; O'Brien, J S

1993-01-01

The effect of saposins (A, B, C and D) on acid sphingomyelinase activity was determined using a crude human kidney sphingomyelinase preparation and a purified sphingomyelinase preparation from human placenta. Saposin D stimulated the activity of the crude enzyme by increasing its apparent Km and Vmax. values for sphingomyelin hydrolysis. Unlike the crude enzyme, the activity of the purified enzyme was strongly inhibited by saposin D as well as other saposins. Saposin D decreased the apparent Km and Vmax values of purified sphingomyelinase activity. The effects of saposin D on the activity of different sphingomyelinase preparations appear to depend on Triton X-100, which is present in the crude enzyme but not in the purified enzyme. When the detergent was removed from the crude preparation, the effect of saposin D changed from being stimulatory to inhibitory. Conversely, when the detergent is added to the purified enzyme, the effect of saposin D on sphingomyelinase activity changed from being inhibitory to stimulatory. While other saposins were inhibitory or had no effect on sphingomyelinase activity in the above assay system, not only saposin D but also saposins A and C exhibited a stimulatory effect upon purified sphingomyelinase activity when the substrate, sphingomyelin, was added in the form of liposomes without detergent. Saposin B was not only inhibitory in the liposome system, but also reduced the stimulatory effect of saposins A, C and D. These observations indicate that the stimulatory effect of saposins A, C and D on acid sphingomyelinase activity is greatly influenced by the physical environment of the enzyme and suggest that similar effects by saposins may be exerted in lysosomal membranes. PMID:8452527
Purification, identification and molecular mechanism of two dipeptidyl peptidase IV (DPP-IV) inhibitory peptides from Antarctic krill (Euphausia superba) protein hydrolysate.

PubMed

Ji, Wei; Zhang, Chaohua; Ji, Hongwu

2017-10-01

Dipeptidyl peptidase IV (DPP-IV) played an important role in blood glucose regulation. Inhibition of DPP-IV may improve glycemic control in diabetics by preventing the rapid breakdown of incretin hormones and prolonging their physiological action. In this study, Antarctic krill (Euphausia superba) protein was hydrolyzed using animal proteolytic enzymes. The hydrolysate was purified sequentially by ultrafiltration, gel filtration chromatography and reversed phase high-performance liquid chromatography (RP-HPLC). DPP-IV inhibitory activity of the fractions achieved from Antarctic krill protein was determined by DPP-IV screening reagent kit. Two purified peptides were identified by Xevo G2-XS QTof mass spectrometer (QTOF-MS). One peptide purified was Ala-Pro (AP) with IC 50 values of 0.0530mg/mL, the other Ile-Pro-Ala (IPA) with IC 50 values of 0.0370mg/mL. They both exhibited strong DPP-IV inhibitory activity. The molecular docking analysis revealed that DPP-IV inhibition by AP and IPA was mainly due to formation of a strong interaction surface force with the 91-96 and 101-105 amino acids of the DPP-IV. Our results suggested that the protein hydrolysate from Antarctic krill can be considered as a promising natural source of DPP-IV inhibitory peptides in the management of diabetes. Copyright © 2017 Elsevier B.V. All rights reserved.
Population mitogenomics provides insights into evolutionary history, source of invasions and diversifying selection in the House Crow (Corvus splendens).

PubMed

Krzemińska, Urszula; Morales, Hernán E; Greening, Chris; Nyári, Árpád S; Wilson, Robyn; Song, Beng Kah; Austin, Christopher M; Sunnucks, Paul; Pavlova, Alexandra; Rahman, Sadequr

2018-04-01

The House Crow (Corvus splendens) is a useful study system for investigating the genetic basis of adaptations underpinning successful range expansion. The species originates from the Indian subcontinent, but has successfully spread through a variety of thermal environments across Asia, Africa and Europe. Here, population mitogenomics was used to investigate the colonisation history and to test for signals of molecular selection on the mitochondrial genome. We sequenced the mitogenomes of 89 House Crows spanning four native and five invasive populations. A Bayesian dated phylogeny, based on the 13 mitochondrial protein-coding genes, supports a mid-Pleistocene (~630,000 years ago) divergence between the most distant genetic lineages. Phylogeographic patterns suggest that northern South Asia is the likely centre of origin for the species. Codon-based analyses of selection and assessments of changes in amino acid properties provide evidence of positive selection on the ND2 and ND5 genes against a background of purifying selection across the mitogenome. Protein homology modelling suggests that four amino acid substitutions inferred to be under positive selection may modulate coupling efficiency and proton translocation mediated by OXPHOS complex I. The identified substitutions are found within native House Crow lineages and ecological niche modelling predicts suitable climatic areas for the establishment of crow populations within the invasive range. Mitogenomic patterns in the invasive range of the species are more strongly associated with introduction history than climate. We speculate that invasions of the House Crow have been facilitated by standing genetic variation that accumulated due to diversifying selection within the native range.
Selective Constraints on Coding Sequences of Nervous System Genes Are a Major Determinant of Duplicate Gene Retention in Vertebrates

PubMed Central

Roux, Julien; Liu, Jialin; Robinson-Rechavi, Marc

2017-01-01

Abstract The evolutionary history of vertebrates is marked by three ancient whole-genome duplications: two successive rounds in the ancestor of vertebrates, and a third one specific to teleost fishes. Biased loss of most duplicates enriched the genome for specific genes, such as slow evolving genes, but this selective retention process is not well understood. To understand what drives the long-term preservation of duplicate genes, we characterized duplicated genes in terms of their expression patterns. We used a new method of expression enrichment analysis, TopAnat, applied to in situ hybridization data from thousands of genes from zebrafish and mouse. We showed that the presence of expression in the nervous system is a good predictor of a higher rate of retention of duplicate genes after whole-genome duplication. Further analyses suggest that purifying selection against the toxic effects of misfolded or misinteracting proteins, which is particularly strong in nonrenewing neural tissues, likely constrains the evolution of coding sequences of nervous system genes, leading indirectly to the preservation of duplicate genes after whole-genome duplication. Whole-genome duplications thus greatly contributed to the expansion of the toolkit of genes available for the evolution of profound novelties of the nervous system at the base of the vertebrate radiation. PMID:28981708
Cellular cooperation in lymphocyte activation. III. B-cell helper effect in the enhancement of T-cell response.

PubMed

Kasahara, T; Kin, K; Itoh, Y; Kawai, T; Kano, Y; Shioiri-Nakano, K

1979-01-01

T and B cells were purified from human tonsil and peripheral blood by the removal of phagocytic cells, followed by filtration through a nylon fiber column (NC) and E-rosette formation. Purified T and B cells contained less than 1% of other cell types. The responses of T cells to concanavalin A (Con A) and soluble protein A were greatly enhanced in the presence of autologous B cells. Participation of B cells in T-cell enhancement was confirmed by the following observations: (a) purified B copulation, which was separated further from adherent B cells, retained its enhancing activity. (b) Another adherent cell-free B-cell preparation, which was purified from the NC-passed fraction, and (c) no T lymphoid but some B lymphoid cell lines, elicited strong T-cell enhancement. It was also found that the enhancing capacity of B cells required no metabolic activity, but rather an intact cell form and direct cell-to-cell contact with responding cells. The stimulatory determinants on B cells were resistant to trypsin and neuraminidase treatment. In this paper a hypothesis will be presented that at least two signals are prerequisite for the effective activation of T cells.
Age estimation based on aspartic acid racemization in elastin from the yellow ligaments.

PubMed

Ritz-Timme, S; Laumeier, I; Collins, M

2003-04-01

The yellow ligaments of the spine are characterized by an exceptionally high content of elastin, a protein with a proved longevity in several human tissues. This unique biochemical composition suggested a suitability of yellow ligaments for age estimation based on aspartic acid racemization (AAR), which was tested by determination of AAR in total tissue specimens and in purified elastin from yellow ligaments of individuals of known age. AAR was found to increase with age in both sample sets. The purified elastin samples exhibited a much faster kinetics than the total tissue, with ca. 3.7-4.6-fold higher apparent rates. The relationship between AAR and age was much closer in the purified elastin samples ( r=0.96-0.99) and it can therefore be used as a basis for biochemical age estimation. The analysis of total tissue samples cannot be recommended since the AAR values can be strongly influenced even by slight, histologically non-detectable variations in the collagen content. Age estimation based on AAR in purified elastin from yellow ligaments may be a valuable additional tool in the identification of unidentified cadavers, especially in cases where other methods cannot be applied (e.g. no available teeth, body parts).
Purification and characterization of a strong fibrinolytic enzyme (nattokinase) in the vegetable cheese natto, a popular soybean fermented food in Japan.

PubMed

Fujita, M; Nomura, K; Hong, K; Ito, Y; Asada, A; Nishimuro, S

1993-12-30

A strong fibrinolytic enzyme (nattokinase) was purified from the vegetable cheese natto. Nattokinase was extracted from natto with saline and isolated by sequential use of hydrophobic chromatography on Butyl-Toyopearl, ion-exchange chromatography on CM-Toyopearl, and gel-filtration on Sephadex G-50. The isolated protein gave a single sharp band on SDS-PAGE either before or after reduction. The sequence, as determined by automated Edman degradation of the uncleaved molecule and its enzymatically derived peptide, consisted of a total 275 amino acid residues (M.W = 27,728) and exhibited a high homology with the subtilisins. The purified nattokinase digested not only fibrin but also several synthetic substrates. Among the synthetic substrates, the most sensitive substrate was Suc-Ala-Ala-Pro-Phe-pNA for subtilisin. PMSF inhibited both the fibrinolytic activity and the amidolytic activity. The results indicate that nattokinase is a subtilisin-like serine protease.
High-throughput protein concentration and buffer exchange: comparison of ultrafiltration and ammonium sulfate precipitation.

PubMed

Moore, Priscilla A; Kery, Vladimir

2009-01-01

High-throughput protein purification is a complex, multi-step process. There are several technical challenges in the course of this process that are not experienced when purifying a single protein. Among the most challenging are the high-throughput protein concentration and buffer exchange, which are not only labor-intensive but can also result in significant losses of purified proteins. We describe two methods of high-throughput protein concentration and buffer exchange: one using ammonium sulfate precipitation and one using micro-concentrating devices based on membrane ultrafiltration. We evaluated the efficiency of both methods on a set of 18 randomly selected purified proteins from Shewanella oneidensis. While both methods provide similar yield and efficiency, the ammonium sulfate precipitation is much less labor intensive and time consuming than the ultrafiltration.
Similar Efficacies of Selection Shape Mitochondrial and Nuclear Genes in Both Drosophila melanogaster and Homo sapiens.

PubMed

Cooper, Brandon S; Burrus, Chad R; Ji, Chao; Hahn, Matthew W; Montooth, Kristi L

2015-08-21

Deleterious mutations contribute to polymorphism even when selection effectively prevents their fixation. The efficacy of selection in removing deleterious mitochondrial mutations from populations depends on the effective population size (Ne) of the mitochondrial DNA and the degree to which a lack of recombination magnifies the effects of linked selection. Using complete mitochondrial genomes from Drosophila melanogaster and nuclear data available from the same samples, we reexamine the hypothesis that nonrecombining animal mitochondrial DNA harbor an excess of deleterious polymorphisms relative to the nuclear genome. We find no evidence of recombination in the mitochondrial genome, and the much-reduced level of mitochondrial synonymous polymorphism relative to nuclear genes is consistent with a reduction in Ne. Nevertheless, we find that the neutrality index, a measure of the excess of nonsynonymous polymorphism relative to the neutral expectation, is only weakly significantly different between mitochondrial and nuclear loci. This difference is likely the result of the larger proportion of beneficial mutations in X-linked relative to autosomal loci, and we find little to no difference between mitochondrial and autosomal neutrality indices. Reanalysis of published data from Homo sapiens reveals a similar lack of a difference between the two genomes, although previous studies have suggested a strong difference in both species. Thus, despite a smaller Ne, mitochondrial loci of both flies and humans appear to experience similar efficacies of purifying selection as do loci in the recombining nuclear genome. Copyright © 2015 Cooper et al.
Transposon Insertions, Structural Variations, and SNPs Contribute to the Evolution of the Melon Genome.

PubMed

Sanseverino, Walter; Hénaff, Elizabeth; Vives, Cristina; Pinosio, Sara; Burgos-Paz, William; Morgante, Michele; Ramos-Onsins, Sebastián E; Garcia-Mas, Jordi; Casacuberta, Josep Maria

2015-10-01

The availability of extensive databases of crop genome sequences should allow analysis of crop variability at an unprecedented scale, which should have an important impact in plant breeding. However, up to now the analysis of genetic variability at the whole-genome scale has been mainly restricted to single nucleotide polymorphisms (SNPs). This is a strong limitation as structural variation (SV) and transposon insertion polymorphisms are frequent in plant species and have had an important mutational role in crop domestication and breeding. Here, we present the first comprehensive analysis of melon genetic diversity, which includes a detailed analysis of SNPs, SV, and transposon insertion polymorphisms. The variability found among seven melon varieties representing the species diversity and including wild accessions and highly breed lines, is relatively high due in part to the marked divergence of some lineages. The diversity is distributed nonuniformly across the genome, being lower at the extremes of the chromosomes and higher in the pericentromeric regions, which is compatible with the effect of purifying selection and recombination forces over functional regions. Additionally, this variability is greatly reduced among elite varieties, probably due to selection during breeding. We have found some chromosomal regions showing a high differentiation of the elite varieties versus the rest, which could be considered as strongly selected candidate regions. Our data also suggest that transposons and SV may be at the origin of an important fraction of the variability in melon, which highlights the importance of analyzing all types of genetic variability to understand crop genome evolution. © The Author 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.
Selection of pecan shell based activated carbons for removal of organic and inorganic impurities from simulated well-water

USDA-ARS?s Scientific Manuscript database

Activated carbons are a byproduct from pyrolysis and have value as a purifying agent. The effectiveness of activated carbons is dependent on feedstock selection and pyrolysis conditions that modify its surface properties. Therefore, pecan shell-based activated carbons (PSACs) were prepared by soakin...
Automated high throughput microscale antibody purification workflows for accelerating antibody discovery

PubMed Central

Luan, Peng; Lee, Sophia; Paluch, Maciej; Kansopon, Joe; Viajar, Sharon; Begum, Zahira; Chiang, Nancy; Nakamura, Gerald; Hass, Philip E.; Wong, Athena W.; Lazar, Greg A.

2018-01-01

ABSTRACT To rapidly find “best-in-class” antibody therapeutics, it has become essential to develop high throughput (HTP) processes that allow rapid assessment of antibodies for functional and molecular properties. Consequently, it is critical to have access to sufficient amounts of high quality antibody, to carry out accurate and quantitative characterization. We have developed automated workflows using liquid handling systems to conduct affinity-based purification either in batch or tip column mode. Here, we demonstrate the capability to purify >2000 antibodies per day from microscale (1 mL) cultures. Our optimized, automated process for human IgG1 purification using MabSelect SuRe resin achieves ∼70% recovery over a wide range of antibody loads, up to 500 µg. This HTP process works well for hybridoma-derived antibodies that can be purified by MabSelect SuRe resin. For rat IgG2a, which is often encountered in hybridoma cultures and is challenging to purify via an HTP process, we established automated purification with GammaBind Plus resin. Using these HTP purification processes, we can efficiently recover sufficient amounts of antibodies from mammalian transient or hybridoma cultures with quality comparable to conventional column purification. PMID:29494273
Variability of selected nutrients and contaminants monitored in rodent diets: A 6-year study

DOE Office of Scientific and Technical Information (OSTI.GOV)

Oller, W.L.; Kendall, D.C.; Greenman, D.L.

1989-01-01

The results are given from monitoring a commercial closed-formula cereal-based rodent diet (Purina 5010), two open-formula cereal-based diets (NIH-31 and NIH-07), and one purified diet (AIN-76) for selected nutrients and contaminants. The observed concentrations of nutrients (protein, fat, vitamin A, and thiamine) approximated the manufacturer specifications for closed-formula cereal diet, while the average concentrations of nutrients found in the open-formula cereal diets were well above the nominal concentrations. Nominal concentrations for these open-formula diets tended to be close to the minimum values that were observed. Except for protein levels, greater variation in nutrient concentrations was found in the purified dietmore » than in the cereal diets. Contaminants were generally much lower in the purified diet than in the cereal diets, but the variation of contaminants was about equal in the two types of diets. Open- and closed-formula cereal diets appear to be very similar to each other in the degree of variation of nutrients and contaminants. Cadmium, lead, and selenium are the constituents of greatest concern in assuring the quality of the rodent diets that were evaluated.« less
A widespread class of reverse transcriptase-related cellular genes.

PubMed

Gladyshev, Eugene A; Arkhipova, Irina R

2011-12-20

Reverse transcriptases (RTs) polymerize DNA on RNA templates. They fall into several structurally related but distinct classes and form an assemblage of RT-like enzymes that, in addition to RTs, also includes certain viral RNA-dependent RNA polymerases (RdRP) synthesizing RNA on RNA templates. It is generally believed that most RT-like enzymes originate from retrotransposons or viruses and have no specific function in the host cell, with telomerases being the only notable exception. Here we report on the discovery and properties of a unique class of RT-related cellular genes collectively named rvt. We present evidence that rvts are not components of retrotransposons or viruses, but single-copy genes with a characteristic domain structure that may contain introns in evolutionarily conserved positions, occur in syntenic regions, and evolve under purifying selection. These genes can be found in all major taxonomic groups including protists, fungi, animals, plants, and even bacteria, although they exhibit patchy phylogenetic distribution in each kingdom. We also show that the RVT protein purified from one of its natural hosts, Neurospora crassa, exists in a multimeric form and has the ability to polymerize NTPs as well as dNTPs in vitro, with a strong preference for NTPs, using Mn(2+) as a cofactor. The existence of a previously unknown class of single-copy RT-related genes calls for reevaluation of the current views on evolution and functional roles of RNA-dependent polymerases in living cells.

Development of a method for the purification and culture of rodent astrocytes.

PubMed

Foo, Lynette C; Allen, Nicola J; Bushong, Eric A; Ventura, P Britten; Chung, Won-Suk; Zhou, Lu; Cahoy, John D; Daneman, Richard; Zong, Hui; Ellisman, Mark H; Barres, Ben A

2011-09-08

The inability to purify and culture astrocytes has long hindered studies of their function. Whereas astrocyte progenitor cells can be cultured from neonatal brain, culture of mature astrocytes from postnatal brain has not been possible. Here, we report a new method to prospectively purify astrocytes by immunopanning. These astrocytes undergo apoptosis in culture, but vascular cells and HBEGF promote their survival in serum-free culture. We found that some developing astrocytes normally undergo apoptosis in vivo and that the vast majority of astrocytes contact blood vessels, suggesting that astrocytes are matched to blood vessels by competing for vascular-derived trophic factors such as HBEGF. Compared to traditional astrocyte cultures, the gene profiles of the cultured purified postnatal astrocytes much more closely resemble those of in vivo astrocytes. Although these astrocytes strongly promote synapse formation and function, they do not secrete glutamate in response to stimulation. Copyright © 2011 Elsevier Inc. All rights reserved.
Immunolocalization of betaine aldehyde dehydrogenase in porcine kidney.

PubMed

Figueroa-Soto, C G; Lopez-Cervantes, G; Valenzuela-Soto, E M

1999-05-19

Polyclonal anti-BADH serum was raised in rabbits against native BADH purified from porcine kidney. The antiserum cross-reacted strongly with BADH purified from kidney, Amaranthus palmierii, and Pseudomona aeuroginosa (1:1000), and weakly with Amaranthus hypochondriacus L (1:100). Antibodies bound to purified native kidney BADH in immunoblots showed a major band of an apparent molecular mass of 340 kDa and a subunit with an apparent molecular mass of 52 kDa. Data on activity assays showed higher activity in cortex sections (81.3 nmol/min/mg protein) than in medulla sections (21.3 nmol/min/mg protein). Immunolocalization of BADH in kidney tissue sections showed that BADH is found in cortex and medulla. In inner medulla, the enzyme was mainly localized in cells surrounding the tubules. Western blot analysis on extracts from the cortex and medulla sections showed higher concentration of BADH protein in cortex than in medulla. These results were in accordance with immunolocalization and activity analysis. Copyright 1999 Academic Press.
Innate Immunity in Lobsters: Partial Purification and Characterization of a Panulirus cygnus Anti-A Lectin.

PubMed

Flower, Robert L P

2012-01-01

A lectin detected in haemolymph from the Australian spiny lobster Panulirus cygnus agglutinated human ABO Group A cells to a higher titre than Group O or B. The lectin also agglutinated rat and sheep erythrocytes, with reactivity with rat erythrocytes strongly enhanced by treatment with the proteolytic enzyme papain, an observation consistent with reactivity via a glycolipid. The lectin, purified by affinity chromatography on fixed rat-erythrocyte stroma, was inhibited equally by N-acetylglucosamine and N-acetylgalactosamine. Comparison of data from gel filtration of haemolymph (behaving as a 1,800,000 Da macromolecule), and polyacrylamide gel electrophoresis of purified lectin (a single 67,000 Da band), suggested that in haemolymph the lecin was a multimer. The purified anti-A lectin autoprecipitated unless the storage solution contained chaotropic inhibitors (125 mmol/L sucrose: 500 mmol/L urea). The properties of this anti-A lectin and other similar lectins are consistent with a role in innate immunity in these invertebrates.
Comprehensive Analysis of a Vibrio parahaemolyticus Strain Extracellular Serine Protease VpSP37

PubMed Central

Bennici, Carmelo; Quatrini, Paola; Catania, Valentina; Mazzola, Salvatore; Ghersi, Giulio; Cuttitta, Angela

2015-01-01

Proteases play an important role in the field of tissue dissociation combined with regenerative medicine. During the years new sources of proteolytic enzymes have been studied including proteases from different marine organisms both eukaryotic and prokaryotic. Herein we have purified a secreted component of an isolate of Vibrio parahaemolyticus, with electrophoretic mobilities corresponding to 36 kDa, belonging to the serine proteases family. Sequencing of the N-terminus enabled the in silico identification of the whole primary structure consisting of 345 amino acid residues with a calculated molecular mass of 37.4 KDa. The purified enzyme, named VpSP37, contains a Serine protease domain between residues 35 and 276 and a canonical Trypsin/Chimotrypsin 3D structure. Functional assays were performed to evaluate protease activity of purified enzyme. Additionally the performance of VpSP37 was evaluated in tissue dissociations experiments and the use of such enzyme as a component of enzyme blend for tissue dissociation procedures is strongly recommended. PMID:26162075
Purification and characterization of a fibrinolytic enzyme from tempeh bongkrek as an alternative of thrombolytic agents

NASA Astrophysics Data System (ADS)

Sasmita, I. R. A.; Sutrisno, A.; Zubaidah, E.; Wardani, A. K.

2018-03-01

Tempeh is one of Indonesia’s traditional foods that contain fibrinolytic enzymes. Tempeh bongkrek shows very strong activity among various tempeh. The fibrinolytic enzymes of bongkrek tempeh are obtained by steps of purification i.e, ammonium sulphate precipitation, ion exchange chromatography and gel filtration chromatography. The fibrinolytic enzymes has been successfully purified with a yield of 4.37%, specific activity of 3,361 U / mg and purification fold of 44.02. SDS PAGE analysis showed that the enzyme was purified in to single band with estimated molecular mass of 75.82 kDa. The purified enzyme has optimum pH of 7 and optimum temperature of 50°C and pH stability between pH 4 - 7 with temperature stability from 30°-50°C. The fibrinolytic activity is increased with addition of CaCl2 but inhibited with CuSO4, phenylmethylsulfonyl fluoride (PMSF), sodium dodecyl sulfate (SDS), and ethylenediaminetetraacetic acid (EDTA).
First isolation of a novel thermostable antifungal peptide secreted by Aspergillus clavatus.

PubMed

Skouri-Gargouri, Houda; Gargouri, Ali

2008-11-01

A novel antifungal peptide produced by an indigenous fungal strain (VR) of Aspergillus clavatus was purified. The antifungal peptide was enriched in the supernatant after heat treatment at 70 degrees C. The thermostable character was exploited in the first purification step, as purified peptide was obtained after ultrafiltration and reverse phase-HPLC on C18 column application. The purified peptide named "AcAFP" for A. clavatus antifungal peptide, has molecular mass of 5773Da determined by MALDI-ToF spectrometry. The N-terminal sequence showed a notable identity to the limited family of antifungal peptides produced by ascomycetes fungi. The AcAFP activity remains intact even after heat treatment at 100 degrees C for 1h confirming its thermostability. It exhibits a strong inhibitory activity against mycelial growth of several serious human and plant pathogenic fungi: Fusariuym oxysporum, Fusarium solani, Aspergillus niger, Botrytis cinerea, Alternaria solani, whereas AcAFP did not affect yeast and bacterial growth.
Evolutionary characterization of hemagglutinin gene of H9N2 influenza viruses isolated from Asia.

PubMed

Shahsavandi, Shahla; Salmanian, Ali-Hatef; Ghorashi, Seyed Ali; Masoudi, Shahin; Ebrahimi, Mohammad Majid

2012-08-01

The full length hemagglutinin (HA) genes of 287 H9N2 AI strains isolated from chickens in Asia during the period 1994-2009 were genetically analyzed. Phylogenetic analysis showed that G1-like viruses circulated in the Middle East and Indian sub-continent countries, whereas other sublineages existed in Far East countries. It also revealed G1-like viruses with an average 96.7% identity clustered into two subgroups largely based on their time of isolation. The Ka/Ks ratio was calculated 0.34 for subgroup 1 and 0.57 for subgroup 2 indicates purifying/stabilizing selection, but despite this there is evidence of localized positive selection when comparing the subgroups 1 and 2 protein sequences. Five sites in HA H9N2 viruses had a posterior probability >0.5 using the Bayesian method, indicating these sites were under positive selection. These sites were found to be associated with the globular head region of HA. To identify sites under positive selection; amino acid substitution classified depends on their radicalism and neutrality. The results indicate that, although most positions in HAs were under purifying selection and can be eliminated, a few positions located in the antigenic regions and receptor binding sites were subject to positive selection. Copyright © 2011 Elsevier Ltd. All rights reserved.
Purification and characterization of acid trehalase from the yeast suc2 mutant.

PubMed

Mittenbühler, K; Holzer, H

1988-06-15

Acid trehalase was purified from the yeast suc2 deletion mutant. After hydrophobic interaction chromatography, the enzyme could be purified to a single band or peak by a further step of either polyacrylamide gel electrophoresis, gel filtration, or isoelectric focusing. An apparent molecular mass of 218,000 Da was calculated from gel filtration. Polyacrylamide gel electrophoresis of the purified enzyme in the presence of sodium dodecyl sulfate suggested a molecular mass of 216,000 Da. Endoglycosidase H digestion of the purified enzyme resulted after sodium dodecyl sulfate gel electrophoresis in one distinct band at 41,000 Da, representing the mannose-free protein moiety of acid trehalase. The carbohydrate content of the enzyme was 86%. Amino acid analysis indicated 354 residues/molecule of enzyme including 9 cysteine moieties and only 1 methionine. The isoelectric point of the enzyme was estimated by gel electrofocusing to be approximately 4.7. The catalytic activity showed a maximum at pH 4.5. The activity of the enzyme was not inhibited by 10 mM each of HgCl2, EDTA, iodoacetic acid, phenanthrolinium chloride or phenylmethylsulfonyl fluoride. There was no activation by divalent metal ions. The acid trehalase exhibited an apparent Km for trehalose of 4.7 +/- 0.1 mM and a Vmax of 99 mumol of trehalose min-1 X mg-1 at 37 degrees C and pH 4.5. The acid trehalase is located in the vacuoles. The rabbit antiserum raised against acid trehalase exhibited strong cross-reaction with purified invertase. These cross-reactions were removed by affinity chromatography using invertase coupled to CNBr-activated Sepharose 4B. Precipitation of acid trehalase activity was observed with the purified antiserum.
Process for removal of water and silicon mu-oxides from chlorosilanes

DOEpatents

Tom, Glenn M.; McManus, James V.

1992-03-10

A scavenger composition having utility for removal of water and silicon mu-oxide impurities from chlorosilanes, such scavenger composition comprising: (a) a support; and (b) associated with the support, one or more compound(s) selected from the group consisting of compounds of the formula: R.sub.a-x MCl.sub.x wherein: M is a metal selected from the group consisting of the monovalent metals lithium, sodium, and potassium; the divalent metals magnesium, strontium, barium, and calcium; and the trivalent metal aluminum; R is alkyl; a is a number equal to the valency of metal M; and x is a number having a value of from 0 to a, inclusive; and wherein said compound(s) of the formula R.sub.a-x MCl.sub.x have been activated for impurity-removal service by a reaction scheme selected from those of the group consisting of: (i) reaction of such compound(s) with hydrogen chloride to form a first reaction product therefrom, followed by reaction of the first reaction product with a chlorosilane of the formula: SiH.sub.4-y Cl.sub.y, wherein y is a number having a value of from 1 to 3, inclusive; and (ii) reaction of such compound(s) with a chlorosilane of the formula: SiH.sub.4-y Cl.sub.y wherein y is a number having a value of 1 to 3, inclusive. A corresponding method of making the scavenger composition, and of purifying a chlorosilane which contains oxygen and silicon mu-oxide impurities, likewise are disclosed, together with a purifier apparatus, in which a bed of the scavenger composition is disposed. The composition, purification process, and purifier apparatus of the invention have utility in purifying gaseous chlorosilanes which are employed in the semiconductor industry as silicon source reagents for forming epitaxial silicon layers.
Composition, process, and apparatus, for removal of water and silicon mu-oxides from chlorosilanes

DOEpatents

Tom, Glenn M.; McManus, James V.

1991-10-15

A scavenger composition having utility for removal of water and silicon mu-oxide impurities from chlorosilanes, such scavenger composition comprising: (a) a support; and (b) associated with the support, one or more compound(s) selected from the group consisting of compounds of the formula: R.sub.a-x MCl.sub.x wherein: M is a metal selected from the group consisting of the monovalent metals lithium, sodium, and potassium; the divalent metals magnesium, strontium, barium, and calcium; and the trivalent metal aluminum; R is alkyl; a is a number equal to the valency of metal M; and x is a number having a value from 0 to a, inclusive; and wherein said compound(s) of the formula R.sub.a-x MCl.sub.x have been activated for impurity-removal service by a reaction scheme selected from those of the group consisting of: (i) reaction of such compound(s) with hydrogen chloride to form a first reaction product therefrom, followed by reaction of the first reaction product with a chlorosilane of the formula: SiH.sub.4"y Cl.sub.y, wherein y is a number having a value of from 1 to 3, inclusive; and (ii) reaction of such compound(s) with a chlorosilane of the formula: SiH.sub.4-y Cl.sub.y wherein y is a number having a value of 1 to 3, inclusive. A corresponding method of making the scavenger composition, and of purifying a chlorosilane which contains oxygen and silicon mu-oxide impurities, likewise are disclosed, together with a purifier apparatus, in which a bed of the scavenger composition is disposed. The composition, purification process, and purifier apparatus of the invention have utility in purifying gaseous chlorosilanes which are employed in the semiconductor industry as silicon source reagents for forming epitaxial silicon layers.
Distance from sub-Saharan Africa predicts mutational load in diverse human genomes.

PubMed

Henn, Brenna M; Botigué, Laura R; Peischl, Stephan; Dupanloup, Isabelle; Lipatov, Mikhail; Maples, Brian K; Martin, Alicia R; Musharoff, Shaila; Cann, Howard; Snyder, Michael P; Excoffier, Laurent; Kidd, Jeffrey M; Bustamante, Carlos D

2016-01-26

The Out-of-Africa (OOA) dispersal ∼ 50,000 y ago is characterized by a series of founder events as modern humans expanded into multiple continents. Population genetics theory predicts an increase of mutational load in populations undergoing serial founder effects during range expansions. To test this hypothesis, we have sequenced full genomes and high-coverage exomes from seven geographically divergent human populations from Namibia, Congo, Algeria, Pakistan, Cambodia, Siberia, and Mexico. We find that individual genomes vary modestly in the overall number of predicted deleterious alleles. We show via spatially explicit simulations that the observed distribution of deleterious allele frequencies is consistent with the OOA dispersal, particularly under a model where deleterious mutations are recessive. We conclude that there is a strong signal of purifying selection at conserved genomic positions within Africa, but that many predicted deleterious mutations have evolved as if they were neutral during the expansion out of Africa. Under a model where selection is inversely related to dominance, we show that OOA populations are likely to have a higher mutation load due to increased allele frequencies of nearly neutral variants that are recessive or partially recessive.
Systems, compositions, and methods for fluid purification

DOEpatents

Ho, W.S. Winston; Verweij, Hendrik; Shqau, Krenar; Ramasubranian, Kartik

2015-12-22

Disclosed herein are membranes comprising a substrate, a support layer, and a selective layer. In some embodiments the membrane may further comprise a permeable layer. Methods of forming membranes are also disclosed comprising forming a support layer on a substrate, removing adsorbed species from the support layer, preparing a solution containing inorganic materials of a selective layer, contacting the support layer with the solution, drying the membrane, and exposing the membrane to rapid thermal processing. Also disclosed are methods of fluid purification comprising providing a membrane having a feed side and a permeable side, passing a fluid mixture across the feed side of the membrane, providing a driving force for transmembrane permeation, removing from the permeate side a permeate stream enriched in a purified fluid, and withdrawing from the feed side a fluid that is depleted in a purified fluid.
Purified phenolics from hydrothermal treatments of biomass: ability to protect sunflower bulk oil and model food emulsions from oxidation.

PubMed

Conde, Enma; Moure, Andrés; Domínguez, Herminia; Gordon, Michael H; Parajó, Juan Carlos

2011-09-14

The phenolic fractions released during hydrothermal treatment of selected feedstocks (corn cobs, eucalypt wood chips, almond shells, chestnut burs, and white grape pomace) were selectively recovered by extraction with ethyl acetate and washed with ethanol/water solutions. The crude extracts were purified by a relatively simple adsorption technique using a commercial polymeric, nonionic resin. Utilization of 96% ethanol as eluting agent resulted in 47.0-72.6% phenolic desorption, yielding refined products containing 49-60% w/w phenolics (corresponding to 30-58% enrichment with respect to the crude extracts). The refined extracts produced from grape pomace and from chestnut burs were suitable for protecting bulk oil and oil-in-water and water-in-oil emulsions. A synergistic action with bovine serum albumin in the emulsions was observed.
Bovicin HC5 inhibits wasteful amino acid degradation by mixed ruminal bacteria in vitro.

PubMed

Lima, Janaína R; Ribon, Andréa de O Barros; Russell, James B; Mantovani, Hilário C

2009-03-01

Streptococcus bovis HC5 produces a broad spectrum lantibiotic (bovicin HC5) that inhibits pure cultures of hyper ammonia-producing bacteria (HAB). Experiments were preformed to see if: (1) S. bovis HC5 cells could inhibit the deamination of amino acids by mixed ruminal bacteria taken directly from a cow, (2) semi-purified bovicin was as effective as S. bovis HC5 cells, and 3) semi-purified and the feed additive monensin were affecting the same types of ammonia-producing ruminal bacteria. Because purified and semi-purified bovicin HC5 was as effective as S. bovis HC5 cells, it appeared that bovicin HC5 was penetrating the cell membranes of HAB before it could be degraded by peptidases and proteinases. Mixed ruminal bacteria that were successively transferred and enriched nine times with trypticase did not become significantly more resistant to either bovicin HC5 (50 AU mL(-1)) or monensin (5 microM), and amplified rDNA restriction analysis indicated that bovicin HC5 and monensin appeared to be selecting against the same types of bacteria.
Cellular immune response in MDR-TB patients to different protein expression of MDR and susceptible Mycobacterium tuberculosis: Rv0147, a novel MDR-TB biomarker.

PubMed

Hadizadeh Tasbiti, Alireza; Yari, Shamsi; Siadat, Seyed Davar; Tabarsi, Payam; Saeedfar, Kayvan; Yari, Fatemeh

2018-02-01

Tuberculosis (TB) is a crucial public health problem with prevalence of multidrug resistant (MDR) rising. An accurate TB biomarker is urgently needed to monitor the response to treatment in patients with MDR tuberculosis. To analyze interaction between selected MDR-TB purified protein and immune cells, dendritic cells from MDR-TB patients and healthy subjects were stimulated by 55KDa protein fractions (Rv0147). The purified proteins identified by proteomic techniques (two-dimensional gel electrophoresis, mass spectrometry) and peptide sequences are known to bind a MHC class I alleles which are extracted from the Immune Epitope Database and Analysis Resource database ( www.iedb.org ). T cells were isolated from PBMC by negative selection and cells were cultured in RPMI-1640 at 37 °C and 5% CO 2 . Cell culture was assayed for cytokine IL-10 and INF-γ by ELISA. We found that INF-γ production was significantly (335 ± 35.5 pg/ml, P ˂ 0.05) upregulated after protein candidate (Rv0147) stimulation by dendritic cells from MDR-TB patients, whereas IL-10 production was greatly reduced compared with production in healthy subjects (212 ± 9.94 pg/ml, P ˂ 0.05). In fact, the purified protein, Rv0147, stimulated dendritic cells from MDR-TB patients, failed to produce IL-10 and directly stimulates INF-γ production by T cells. These results suggest that the purified protein, Rv0147, may stimulate Th1 type protective cytokine response in MDR-TB patients but not in normal subjects. The production of INF-γ but not IL-10 in the presence of purified protein, Rv0147, may be shifted to Th1 responses in MDR-TB patients and supports its potential as protein vaccine candidates against TB.
Potent malaria transmission-blocking antibody responses elicited by Plasmodium falciparum Pfs25 expressed in Escherichia coli after successful protein refolding.

PubMed

Kumar, Rajesh; Angov, Evelina; Kumar, Nirbhay

2014-04-01

Production of Pfs25, a Plasmodium falciparum transmission-blocking vaccine target antigen, in functional conformation with the potential to elicit effective immunogenicity still remains a major challenge. In the current study, codon-harmonized recombinant Pfs25 (CHrPfs25) was expressed in Escherichia coli, and purified protein after simple oxidative refolding steps retained reduction-sensitive conformational epitopes of transmission-blocking monoclonal antibodies. CHrPfs25 formulated in several adjuvants elicited strong immunogenicity in preclinical studies in mice. Antibodies elicited after immunization recognized native Pfs25 on the surface of live gametes of P. falciparum and demonstrated complete malaria transmission-blocking activity. The transmission-blocking efficacy was 100% even after a 1:128 dilution of sera from immunized mice in the complete Freund's adjuvant and Montanide ISA51 groups and after a 1:16 dilution of sera from mice in the alum group. The blocking was mediated by antibodies; purified IgG at concentrations as low as 31.25 μg/ml exhibited 100% transmission blocking in membrane feeding assays employing two different species of mosquitoes, Anopheles gambiae and Anopheles stephensi. This study provides the first evidence for successful expression of biologically functional rPfs25 in E. coli. The extremely potent malaria transmission-blocking activity of antibodies elicited by immunization with purified protein provides strong support for further evaluation of E. coli-derived CHrPfs25 as a malaria transmission-blocking vaccine in human clinical trials.
Allergen-specific IgG antibodies purified from mite-allergic patients sera block the IgE recognition of Dermatophagoides pteronyssinus antigens: an in vitro study.

PubMed

Siman, Isabella Lima; de Aquino, Lais Martins; Ynoue, Leandro Hideki; Miranda, Juliana Silva; Pajuaba, Ana Claudia Arantes Marquez; Cunha-Júnior, Jair Pereira; Silva, Deise Aparecida Oliveira; Taketomi, Ernesto Akio

2013-01-01

One of the purposes of specific immunotherapy (SIT) is to modulate humoral immune response against allergens with significant increases in allergen-specific IgG levels, commonly associated with blocking activity. The present study investigated in vitro blocking activity of allergen-specific IgG antibodies on IgE reactivity to Dermatophagoides pteronyssinus (Dpt) in sera from atopic patients. Dpt-specific IgG antibodies were purified by ammonium sulfate precipitation followed by protein-G affinity chromatography. Purity was checked by SDS-PAGE and immunoreactivity by slot-blot and immunoblot assays. The blocking activity was evaluated by inhibition ELISA. The electrophoretic profile of the ammonium sulfate precipitated fraction showed strongly stained bands in ligand fraction after chromatography, compatible with molecular weight of human whole IgG molecule. The purity degree was confirmed by detecting strong immunoreactivity to IgG, negligible to IgA, and no reactivity to IgE and IgM. Dpt-specific IgG fraction was capable of significantly reducing levels of IgE anti-Dpt, resulting in 35%-51% inhibition of IgE reactivity to Dpt in atopic patients sera. This study showed that allergen-specific IgG antibodies purified from mite-allergic patients sera block the IgE recognition of Dermatophagoides pteronyssinus antigens. This approach reinforces that intermittent measurement of serum allergen-specific IgG antibodies will be an important objective laboratorial parameter that will help specialists to follow their patients under SIT.
In vitro production and antifungal activity of peptide ABP-dHC-cecropin A.

PubMed

Zhang, Jiaxin; Movahedi, Ali; Xu, Junjie; Wang, Mengyang; Wu, Xiaolong; Xu, Chen; Yin, Tongming; Zhuge, Qiang

2015-04-10

The antimicrobial peptide ABP-dHC-cecropin A is a small cationic peptide with potent activity against a wide range of bacterial species. Evidence of antifungal activity has also been suggested; however, testing of this peptide has been limited due to the low expression of cecropin proteins in Escherichia coli. To improve expression of this peptide in E. coli, ABP-dHC-cecropin A was cloned into a pSUMO vector and transformed into E. coli, resulting in the production of a pSUMO-ABP-dHC-cecropin A fusion protein. The soluble form of this protein was then purified by Ni-IDA chromatography, yielding a total of 496-mg protein per liter of fermentation culture. The SUMO-ABP-dHC-cecropin A fusion protein was then cleaved using a SUMO protease and re-purified by Ni-IDA chromatography, yielding a total of 158-mg recombinant ABP-dHC-cecropin A per liter of fermentation culture at a purity of ≥94%, the highest yield reported to date. Antifungal activity assays performed using this purified recombinant peptide revealed strong antifungal activity against both Candida albicans and Neurospora crassa, as well as Rhizopus, Fusarium, Alternaria, and Mucor species. Combined with previous analyses demonstrating strong antibacterial activity against a number of important bacterial pathogens, these results confirm the use of ABP-dHC-cecropin A as a broad-spectrum antimicrobial peptide, with significant therapeutic potential. Copyright © 2015 Elsevier B.V. All rights reserved.
Biopotency of serine protease inhibitors from cowpea (Vigna unguiculata) seeds on digestive proteases and the development of Spodoptera littoralis (Boisduval).

PubMed

Abd El-latif, Ashraf Oukasha

2015-05-01

Serine protease inhibitors (PIs) have been described in many plant species and are universal throughout the plant kingdom, where trypsin inhibitors is the most common type. In the present study, trypsin and chymotrypsin inhibitory activity was detected in the seed flour extracts of 13 selected cultivars/accessions of cowpea. Two cowpea cultivars, Cream7 and Buff, were found to have higher trypsin and chymotrypsin inhibitory potential compared to other tested cultivars for which they have been selected for further purification studies using ammonium sulfate fractionation and DEAE-Sephadex A-25 column. Cream7-purified proteins showed two bands on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) corresponding to molecular mass of 17.10 and 14.90 kDa, while the purified protein from Buff cultivar showed a single band corresponding mass of 16.50 kDa. The purified inhibitors were stable at temperature below 60°C and were active at wide range of pH from 2 to 12. The kinetic analysis revealed noncompetitive type of inhibition for both inhibitors against both enzymes. The inhibitor constant (Ki ) values suggested high affinity between inhibitors and enzymes. Purified inhibitors were found to have deep and negative effects on the mean larval weight, larval mortality, pupation, and mean pupal weight of Spodoptera littoralis, where Buff PI was more effective than Cream7 PI. It may be concluded that cowpea PI gene(s) could be potential insect control protein for future studies in developing insect-resistant transgenic plants. © 2014 Wiley Periodicals, Inc.
Mutants of feline immunodeficiency virus resistant to 2',3'-dideoxy-2',3'-didehydrothymidine.

PubMed Central

Zhu, Y Q; Remington, K M; North, T W

1996-01-01

We selected mutants of feline immunodeficiency virus (FIV) that are resistant to 2',3'-dideoxy-2',3'-didehydrothymidine (d4T). Two mutants were selected in cultured cells with a stepwise increase in d4T concentration, resulting in mutants able to replicate in 100 microM d4T. These mutants were three- to sixfold more resistant to d4T than wild-type FIV. They were also cross-resistant to 3'-azido-3'-deoxythymidine (AZT), 3'-fluoro-2',3'-dideoxythymidine, 2',3'-dideoxycytidine, 2',3'-dideoxyinosine, and 9-(2-phosphonylmethoxyethyl)adenine, and they were highly resistant to phosphonoformic acid (PFA). Plaque-purified mutants were isolated from each of the mutant populations. The mutant phenotype was stable, because both of the plaque-purified mutants remained d4T resistant even after three passages in the absence of d4T. One of the plaque-purified mutants, designated D4R-3c, was further characterized. Compared with wild-type reverse transcriptase (RT), RT purified from D4R-3c was 3-fold resistant to inhibition by the 5'-triphosphate of d4T, 10-fold resistant to inhibition by the 5'-triphosphate of AZT, and 6-fold resistant to PFA. D4R-3c had a single point mutation in the RT-encoding region of the pol gene at position 2474, resulting in a Val to Ile mutation at codon 47 of the FIV RT. The role of this mutation in d4T resistance was confirmed by site-directed mutagenesis. PMID:8878567

Purification of diverse hemoglobins by metal salt precipitation.

PubMed

Zimmerman, Devon; Dienes, Jack; Abdulmalik, Osheiza; Elmer, Jacob J

2016-09-01

Although donated blood is the preferred material for transfusion, its limited availability and stringent storage requirements have motivated the development of blood substitutes. The giant extracellular hemoglobin (aka erythrocruorin) of the earthworm Lumbricus terrestris (LtEc) has shown promise as a blood substitute, but an efficient purification method for LtEc must be developed to meet the potential large demand for blood substitutes. In this work, an optimized purification process that uses divalent and trivalent metal salts to selectively precipitate human, earthworm, and bloodworm hemoglobin (HbA, LtEc, and GdHb, respectively) from crude solutions was developed. Although several metal ions were able to selectively precipitate LtEc, Zn(2+) and Ni(2+) provided the lowest heme oxidation and highest overall yield of LtEc. In contrast, Zn(2+) was the only metal ion that completely precipitated HbA and GdHb. Polyacrylamide gel electrophoresis (PAGE) analysis shows that metal precipitation removes several impurities to provide highly pure hemoglobin samples. Heme oxidation levels were relatively low for Zn(2+)-purified HbA and LtEc (2.4±1.3% and 5.3±2.1%, respectively), but slightly higher for Ni(2+)-purified LtEc (8.4±1.2%). The oxygen affinity and cooperativity of the precipitated samples are also identical to samples purified with tangential flow filtration (TFF) alone, indicating the metal precipitation does not significantly affect the function of the hemoglobins. Overall, these results show that hemoglobins from several different species can be highly purified using a combination of metal (Zn(2+)) precipitation and tangential flow filtration. Copyright © 2015 Elsevier Inc. All rights reserved.
Purification and immunogenicity study of human papillomavirus 58 virus-like particles expressed in Pichia pastoris.

PubMed

Jiang, Zijun; Tong, Guangjie; Cai, Beibei; Xu, Yihan; Lou, Jueren

2011-12-01

Two human papillomavirus (HPV) prophylactic vaccines are currently available in the market: Gardasil and Cervarix. These two vaccines work against tumor high-risk subtypes HPV 16 and HPV 18. However, they do not include other high-risk subtypes such as HPV 58. Epidemiological research in China shows that HPV 58 is a prevalent high-risk subtype, second only to HPV 16 and HPV 18. Thus, for cervical cancer prevention in China, developing a vaccine against HPV 58 is necessary. In this study, HPV 58 virus-like particles (VLPs) were expressed in the Pichia pastoris, and subsequently purified through pretreatment and a three-step purification process consisting of strong cation exchange chromatography, size-exclusion chromatography, and hydroxyapatite chromatography. The highly purified HPV 58 VLPs were confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, electron microscopy, dynamic laser scattering, and ultracentrifugation. The purified VLPs were used to immunize mice to test their ability to induce humoral immunity. Enzyme-linked immunosorbent assays were performed on the sera of the immunized mice and significantly high anti-HPV 58 VLP antibody titers were observed. The immunogenicity study demonstrates that the purified HPV 58 VLPs are HPV vaccine candidates. Copyright © 2011 Elsevier Inc. All rights reserved.
A Cytotoxic Antibody Recognizing Lacto-N-fucopentaose I (LNFP I) on Human Induced Pluripotent Stem (hiPS) Cells*

PubMed Central

Matsumoto, Shogo; Nakao, Hiromi; Kawabe, Keiko; Nonaka, Motohiro; Toyoda, Hidenao; Takishima, Yuto; Kawabata, Kenji; Yamaguchi, Tomoko; Furue, Miho K.; Taki, Takao; Okumura, Takeshi; Yamazaki, Yuzo; Nakaya, Shuuichi; Kawasaki, Nobuko; Kawasaki, Toshisuke

2015-01-01

We have generated a mouse monoclonal antibody (R-17F, IgG1 subtype) specific to human induced pluripotent stem (hiPS)/embryonic stem (ES) cells by using a hiPS cell line as an antigen. Triple-color confocal immunostaining images of hiPS cells with R-17F indicated that the R-17F epitope was expressed exclusively and intensively on the cell membranes of hiPS cells and co-localized partially with those of SSEA-4 and SSEA-3. Lines of evidence suggested that the predominant part of the R-17F epitope was a glycolipid. Upon TLC blot of total lipid extracts from hiPS cells with R-17F, one major R-17F-positive band was observed at a slow migration position close to that of anti-blood group H1(O) antigen. MALDI-TOF-MS and MSn analyses of the purified antigen indicated that the presumptive structure of the R-17F antigen was Fuc-Hex-HexNAc-Hex-Hex-Cer. Glycan microarray analysis involving 13 different synthetic oligosaccharides indicated that R-17F bound selectively to LNFP I (Fucα1–2Galβ1–3GlcNAcβ1–3Galβ1–4Glc). A critical role of the terminal Fucα1–2 residue was confirmed by the selective disappearance of R-17F binding to the purified antigen upon α1–2 fucosidase digestion. Most interestingly, R-17F, when added to hiPS/ES cell suspensions, exhibited potent dose-dependent cytotoxicity. The cytotoxic effect was augmented markedly upon the addition of the secondary antibody (goat anti-mouse IgG1 antibody). R-17F may be beneficial for safer regenerative medicine by eliminating residual undifferentiated hiPS cells in hiPS-derived regenerative tissues, which are considered to be a strong risk factor for carcinogenesis. PMID:26100630
Global patterns of diversity and selection in human tyrosinase gene.

PubMed

Hudjashov, Georgi; Villems, Richard; Kivisild, Toomas

2013-01-01

Global variation in skin pigmentation is one of the most striking examples of environmental adaptation in humans. More than two hundred loci have been identified as candidate genes in model organisms and a few tens of these have been found to be significantly associated with human skin pigmentation in genome-wide association studies. However, the evolutionary history of different pigmentation genes is rather complex: some loci have been subjected to strong positive selection, while others evolved under the relaxation of functional constraints in low UV environment. Here we report the results of a global study of the human tyrosinase gene, which is one of the key enzymes in melanin production, to assess the role of its variation in the evolution of skin pigmentation differences among human populations. We observe a higher rate of non-synonymous polymorphisms in the European sample consistent with the relaxation of selective constraints. A similar pattern was previously observed in the MC1R gene and concurs with UV radiation-driven model of skin color evolution by which mutations leading to lower melanin levels and decreased photoprotection are subject to purifying selection at low latitudes while being tolerated or even favored at higher latitudes because they facilitate UV-dependent vitamin D production. Our coalescent date estimates suggest that the non-synonymous variants, which are frequent in Europe and North Africa, are recent and have emerged after the separation of East and West Eurasian populations.
Warning signals are under positive frequency-dependent selection in nature

PubMed Central

Chouteau, Mathieu; Arias, Mónica; Joron, Mathieu

2016-01-01

Positive frequency-dependent selection (FDS) is a selection regime where the fitness of a phenotype increases with its frequency, and it is thought to underlie important adaptive strategies resting on signaling and communication. However, whether and how positive FDS truly operates in nature remains unknown, which hampers our understanding of signal diversity. Here, we test for positive FDS operating on the warning color patterns of chemically defended butterflies forming multiple coexisting mimicry assemblages in the Amazon. Using malleable prey models placed in localities showing differences in the relative frequencies of warningly colored prey, we demonstrate that the efficiency of a warning signal increases steadily with its local frequency in the natural community, up to a threshold where protection stabilizes. The shape of this relationship is consistent with the direct effect of the local abundance of each warning signal on the corresponding avoidance knowledge of the local predator community. This relationship, which differs from purifying selection acting on each mimetic pattern, indicates that predator knowledge, integrated over the entire community, is saturated only for the most common warning signals. In contrast, among the well-established warning signals present in local prey assemblages, most are incompletely known to local predators and enjoy incomplete protection. This incomplete predator knowledge should generate strong benefits to life history traits that enhance warning efficiency by increasing the effective frequency of prey visible to predators. Strategies such as gregariousness or niche convergence between comimics may therefore readily evolve through their effects on predator knowledge and warning efficiency. PMID:26858416
Different Evolutionary Paths to Complexity for Small and Large Populations of Digital Organisms

PubMed Central

2016-01-01

A major aim of evolutionary biology is to explain the respective roles of adaptive versus non-adaptive changes in the evolution of complexity. While selection is certainly responsible for the spread and maintenance of complex phenotypes, this does not automatically imply that strong selection enhances the chance for the emergence of novel traits, that is, the origination of complexity. Population size is one parameter that alters the relative importance of adaptive and non-adaptive processes: as population size decreases, selection weakens and genetic drift grows in importance. Because of this relationship, many theories invoke a role for population size in the evolution of complexity. Such theories are difficult to test empirically because of the time required for the evolution of complexity in biological populations. Here, we used digital experimental evolution to test whether large or small asexual populations tend to evolve greater complexity. We find that both small and large—but not intermediate-sized—populations are favored to evolve larger genomes, which provides the opportunity for subsequent increases in phenotypic complexity. However, small and large populations followed different evolutionary paths towards these novel traits. Small populations evolved larger genomes by fixing slightly deleterious insertions, while large populations fixed rare beneficial insertions that increased genome size. These results demonstrate that genetic drift can lead to the evolution of complexity in small populations and that purifying selection is not powerful enough to prevent the evolution of complexity in large populations. PMID:27923053
Full-genome dengue virus sequencing in mosquito saliva shows lack of convergent positive selection during transmission by Aedes aegypti

PubMed Central

Cao-Lormeau, Van-Mai; Lambrechts, Louis

2017-01-01

Abstract Like other pathogens with high mutation and replication rates, within-host dengue virus (DENV) populations evolve during infection of their main mosquito vector, Aedes aegypti. Within-host DENV evolution during transmission provides opportunities for adaptation and emergence of novel virus variants. Recent studies of DENV genetic diversity failed to detect convergent evolution of adaptive mutations in mosquito tissues such as midgut and salivary glands, suggesting that convergent positive selection is not a major driver of within-host DENV evolution in the vector. However, it is unknown whether this conclusion extends to the transmitted viral subpopulation because it is technically difficult to sequence DENV genomes in mosquito saliva. Here, we achieved DENV full-genome sequencing by pooling saliva samples collected non-sacrificially from 49 to 163 individual Ae. aegypti mosquitoes previously infected with one of two DENV-1 genotypes. We compared the transmitted viral subpopulations found in the pooled saliva samples collected in time series with the input viral population present in the infectious blood meal. In all pooled saliva samples examined, the full-genome consensus sequence of the input viral population was unchanged. Although the pooling strategy prevents analysis of individual saliva samples, our results demonstrate the lack of strong convergent positive selection during a single round of DENV transmission by Ae. aegypti. This finding reinforces the idea that genetic drift and purifying selection are the dominant evolutionary forces shaping within-host DENV genetic diversity during transmission by mosquitoes. PMID:29497564
Purification and characterization of an alkaline protease from Micrococcus sp. isolated from the South China Sea

NASA Astrophysics Data System (ADS)

Hou, Enling; Xia, Tao; Zhang, Zhaohui; Mao, Xiangzhao

2017-04-01

Protease is wildly used in various fields, such as food, medicine, washing, leather, cosmetics and other industrial fields. In this study, an alkaline protease secreted by Micrococcus NH54PC02 isolated from the South China Sea was purified and characterized. The growth curve and enzyme activity curve indicated that the cell reached a maximum concentration at the 30th hour and the enzyme activity reached the maximum value at the 36th hour. The protease was purified with 3 steps involving ammonium sulfate precipitation, ion-exchange chromatography and hydrophobic chromatography with 8.22-fold increase in specific activity and 23.68% increase in the recovery. The molecular mass of the protease was estimated to be 25 kDa by SDS-PAGE analysis. The optimum temperature and pH for the protease activity were 50°C and pH 10.0, respectively. The protease showed a strong stability in a wide range of pH values ranging from 6.0-11.0, and maintained 90% enzyme activity in strong alkaline environment with pH 11.0. Inhibitor trials indicated that the protease might be serine protease. But it also possessed the characteristic of metalloprotease as it could be strongly inhibited by EDTA and strongly stimulated by Mn2+. Evaluation of matrix-assisted laser desorption ionization/time-of-flight MS (MALDI-TOF-TOF/MS) showed that the protease might belong to the peptidase S8 family.
Evolution of the Male-Determining Gene SRY Within the Cat Family Felidae

PubMed Central

King, V.; Goodfellow, P. N.; Wilkerson, A. J. Pearks; Johnson, W. E.; O'Brien, S. J.; Pecon-Slattery, J.

2007-01-01

In most placental mammals, SRY is a single-copy gene located on the Y chromosome and is the trigger for male sex determination during embryonic development. Here, we present comparative genomic analyses of SRY (705 bp) along with the adjacent noncoding 5′ flank (997 bp) and 3′ flank (948 bp) in 36 species of the cat family Felidae. Phylogenetic analyses indicate that the noncoding genomic flanks and SRY closely track species divergence. However, several inconsistencies are observed in SRY. Overall, the gene exhibits purifying selection to maintain function (ω = 0.815) yet SRY is under positive selection in two of the eight felid lineages. SRY has low numbers of nucleotide substitutions, yet most encode amino acid changes between species, and four different species have significantly altered SRY due to insertion/deletions. Moreover, fixation of nonsynonymous substitutions between sister taxa is not consistent and may occur rapidly, as in the case of domestic cat, or not at all over long periods of time, as observed within the Panthera lineage. The former resembles positive selection during speciation, and the latter purifying selection to maintain function. Thus, SRY evolution in cats likely reflects the different phylogeographic histories, selection pressures, and patterns of speciation in modern felids. PMID:17277366
Searching for evidence of selection in avian DNA barcodes.

PubMed

Kerr, Kevin C R

2011-11-01

The barcode of life project has assembled a tremendous number of mitochondrial cytochrome c oxidase I (COI) sequences. Although these sequences were gathered to develop a DNA-based system for species identification, it has been suggested that further biological inferences may also be derived from this wealth of data. Recurrent selective sweeps have been invoked as an evolutionary mechanism to explain limited intraspecific COI diversity, particularly in birds, but this hypothesis has not been formally tested. In this study, I collated COI sequences from previous barcoding studies on birds and tested them for evidence of selection. Using this expanded data set, I re-examined the relationships between intraspecific diversity and interspecific divergence and sampling effort, respectively. I employed the McDonald-Kreitman test to test for neutrality in sequence evolution between closely related pairs of species. Because amino acid sequences were generally constrained between closely related pairs, I also included broader intra-order comparisons to quantify patterns of protein variation in avian COI sequences. Lastly, using 22 published whole mitochondrial genomes, I compared the evolutionary rate of COI against the other 12 protein-coding mitochondrial genes to assess intragenomic variability. I found no conclusive evidence of selective sweeps. Most evidence pointed to an overall trend of strong purifying selection and functional constraint. The COI protein did vary across the class Aves, but to a very limited extent. COI was the least variable gene in the mitochondrial genome, suggesting that other genes might be more informative for probing factors constraining mitochondrial variation within species. © 2011 Blackwell Publishing Ltd.
Quantification of quercetin glycosides in 6 onion cultivars and comparisons of hydrolysis-HPLC and spectrophotometric methods in measuring total quercetin concentrations.

PubMed

Yoo, Kil Sun; Lee, Eun Jin; Patil, Bhimanagouda S

2010-03-01

This study was performed to purify and quantify quercetin glycosides (QG) and aglycone (free) quercetin (Q) in 6 selected onion cultivars and to compare analytical approaches based on high-performance liquid chromatography (HPLC) and spectrophotometry for the quantification of total quercetin (TQ) concentrations. Individual mono- and di-glycoside Q compounds were purified using a semipreparative HPLC and identified by comparing spectral data and by confirming corresponding peaks of QG and Q after incomplete enzyme-hydrolysis. Purified QG were quantified as Q by enzyme-hydrolysis/HPLC. TQ concentrations obtained from 20 onion bulbs with enzyme-hydrolysis/HPLC, no-hydrolysis/HPLC, and a spectrophotometric method without prior hydrolysis were significantly correlated (r(2)= 0.99) and were about 15% higher, identical, or 10% less than those concentrations by a standard acid-hydrolysis/HPLC method, respectively. During enzyme-hydrolysis of onion extracts, progressive reduction of the QG and formation of the corresponding mono-glycosides and Q were monitored using an analytical HPLC. TQ ranged from 83 to 330 microg/g F.W. in 6 selected cultivars of long-day or short-day onions. Q3,4'G and Q4'G were the 2 major compounds and comprised approximately between 94% and 97% of TQ in onions.
Anticancer activity of bacteriophage T4 and its mutant HAP1 in mouse experimental tumour models.

PubMed

Dabrowska, Krystyna; Opolski, Adam; Wietrzyk, Joanna; Switala-Jelen, Kinga; Godlewska, Joanna; Boratynski, Janusz; Syper, Danuta; Weber-Dabrowska, Beata; Gorski, Andrzej

2004-01-01

Previously, we have shown the ability of the bacteriophage T4 and its substrain HAP1 (selected for a higher affinity to melanoma cells) to reveal antimetastatic activity in a mouse melanoma model. Here, we investigated the potential phage anticancer activity in primary tumour models. Mice were inoculated subcutaneously with B16 or LLC cells (collected from in vitro culture). Bacteriophages T4 and HAP1 were injected intraperitoneally daily (8 x 10(8)pfu/mouse, except the experiment concerning the dose-dependence). Treatment with purified preparations of bacteriophage T4 resulted in significant reduction of tumour size, the effect being dose-dependent. HAP1 was more effective than T4 and its activity was also dose-dependent. Parallel experiments with non-purified bacteriophage lysates resulted in significant stimulation of tumour growth. These data suggest that purified bacteriophages may inhibit tumour growth, a phenomenon with potentially important clinical implications in oncology.
Evolution of the Insect Desaturase Gene Family with an Emphasis on Social Hymenoptera

PubMed Central

Helmkampf, Martin; Cash, Elizabeth; Gadau, Jürgen

2015-01-01

Desaturase genes are essential for biological processes, including lipid metabolism, cell signaling, and membrane fluidity regulation. Insect desaturases are particularly interesting for their role in chemical communication, and potential contribution to speciation, symbioses, and sociality. Here, we describe the acyl-CoA desaturase gene families of 15 insects, with a focus on social Hymenoptera. Phylogenetic reconstruction revealed that the insect desaturases represent an ancient gene family characterized by eight subfamilies that differ strongly in their degree of conservation and frequency of gene gain and loss. Analyses of genomic organization showed that five of these subfamilies are represented in a highly microsyntenic region conserved across holometabolous insect taxa, indicating an ancestral expansion during early insect evolution. In three subfamilies, ants exhibit particularly large expansions of genes. Despite these expansions, however, selection analyses showed that desaturase genes in all insect lineages are predominantly undergoing strong purifying selection. Finally, for three expanded subfamilies, we show that ants exhibit variation in gene expression between species, and more importantly, between sexes and castes within species. This suggests functional differentiation of these genes and a role in the regulation of reproductive division of labor in ants. The dynamic pattern of gene gain and loss of acyl-CoA desaturases in ants may reflect changes in response to ecological diversification and an increased demand for chemical signal variability. This may provide an example of how gene family expansions can contribute to lineage-specific adaptations through structural and regulatory changes acting in concert to produce new adaptive phenotypes. PMID:25425561
Selective Constraints on Coding Sequences of Nervous System Genes Are a Major Determinant of Duplicate Gene Retention in Vertebrates.

PubMed

Roux, Julien; Liu, Jialin; Robinson-Rechavi, Marc

2017-11-01

The evolutionary history of vertebrates is marked by three ancient whole-genome duplications: two successive rounds in the ancestor of vertebrates, and a third one specific to teleost fishes. Biased loss of most duplicates enriched the genome for specific genes, such as slow evolving genes, but this selective retention process is not well understood. To understand what drives the long-term preservation of duplicate genes, we characterized duplicated genes in terms of their expression patterns. We used a new method of expression enrichment analysis, TopAnat, applied to in situ hybridization data from thousands of genes from zebrafish and mouse. We showed that the presence of expression in the nervous system is a good predictor of a higher rate of retention of duplicate genes after whole-genome duplication. Further analyses suggest that purifying selection against the toxic effects of misfolded or misinteracting proteins, which is particularly strong in nonrenewing neural tissues, likely constrains the evolution of coding sequences of nervous system genes, leading indirectly to the preservation of duplicate genes after whole-genome duplication. Whole-genome duplications thus greatly contributed to the expansion of the toolkit of genes available for the evolution of profound novelties of the nervous system at the base of the vertebrate radiation. © The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.
Evolution of high mobility group nucleosome-binding proteins and its implications for vertebrate chromatin specialization.

PubMed

González-Romero, Rodrigo; Eirín-López, José M; Ausió, Juan

2015-01-01

High mobility group (HMG)-N proteins are a family of small nonhistone proteins that bind to nucleosomes (N). Despite the amount of information available on their structure and function, there is an almost complete lack of information on the molecular evolutionary mechanisms leading to their exclusive differentiation. In the present work, we provide evidence suggesting that HMGN lineages constitute independent monophyletic groups derived from a common ancestor prior to the diversification of vertebrates. Based on observations of the functional diversification across vertebrate HMGN proteins and on the extensive silent nucleotide divergence, our results suggest that the long-term evolution of HMGNs occurs under strong purifying selection, resulting from the lineage-specific functional constraints of their different protein domains. Selection analyses on independent lineages suggest that their functional specialization was mediated by bursts of adaptive selection at specific evolutionary times, in a small subset of codons with functional relevance-most notably in HMGN1, and in the rapidly evolving HMGN5. This work provides useful information to our understanding of the specialization imparted on chromatin metabolism by HMGNs, especially on the evolutionary mechanisms underlying their functional differentiation in vertebrates. © The Author 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.
Anticoagulant and antithrombotic evaluation of native fucosylated chondroitin sulfates and their derivatives as selective inhibitors of intrinsic factor Xase.

PubMed

Wu, Mingyi; Wen, Dandan; Gao, Na; Xiao, Chuang; Yang, Lian; Xu, Li; Lian, Wu; Peng, Wenlie; Jiang, Jianmin; Zhao, Jinhua

2015-03-06

Fucosylated chondroitin sulfate (FCS), a structurally unusual glycosaminoglycan, has distinct anticoagulant properties, and is an especially strong inhibitor of the intrinsic factor Xase (anti-Xase). To obtain a highly selective inhibitor of human Xase, we purified six native FCSs with various sulfation patterns, prepared a series of FCS derivatives, and then elucidated the relationship between the structures and the anticoagulant activities of FCSs. FCSs 1-3 containing higher Fuc2S4S exhibit stronger AT-dependent anti-IIa activities, whereas 4-6 containing more Fuc3S4S produce potent HCII-dependent anti-IIa activities. Saccharides containing a minimum of 6-8 trisaccharide units, free carboxyl groups, and full fucosylation of GlcA may be required for potent anti-Xase activity, and approximately six trisaccharide units and partial fucosylation of GlcA may contribute to potent HCII-dependent activity. Decreasing of the molecular weights markedly reduces their AT-dependent anti-IIa activities, and even eliminates human platelet and factor XII activation. Furthermore, in vitro and in vivo studies suggested that fractions of 6-12 kDa may be very promising compounds as putative selective intrinsic Xase inhibitors with antithrombotic action, but without the consequences of major bleeding and factor XII activation. Copyright © 2014 Elsevier Masson SAS. All rights reserved.
[Study on extraction and purification technology of Hubei ophiopogon saponins].

PubMed

Lin, Yun-Han; Li, Chong-Ming; Li, Xiao-Dong; Xiang, Yang; Zhang, Ya-Qin; Zhang, Xiao-Cun; Liu, Xia

2013-05-01

To explore the extraction and purification technology of total saponins from the effective parts of Liriope spicata. Orthogonal design was used. Macroporous resin was selected to separate and purify total saponin from the effective parts of Liriope spicata. The process validation was conducted. The total saponins was determined by Ultraviolet Spectrophotometry. The optimal extraction conditions were as follows: 10 times the amount of ethanol (70%) for each occasion and hot reflux (3 x 2 h). Total saponins was purified by D101 macroporous resin. The concentration of eluting ethanol was 50% - 70%. Ethanol (70%) was selected as the best eluent. The result of process validation was consistent with the study. The process is simple and stable enough to significantly improve the extraction rate of the effective parts. The study can provide reference for the research and production of effective parts of traditional Chinese medicine such as Liriope spicata.
Host-Parasite Interactions and Purifying Selection in a Microsporidian Parasite of Honey Bees

PubMed Central

Huang, Qiang; Chen, Yan Ping; Wang, Rui Wu; Cheng, Shang; Evans, Jay D.

2016-01-01

To clarify the mechanisms of Nosema ceranae parasitism, we deep-sequenced both honey bee host and parasite mRNAs throughout a complete 6-day infection cycle. By time-series analysis, 1122 parasite genes were significantly differently expressed during the reproduction cycle, clustering into 4 expression patterns. We found reactive mitochondrial oxygen species modulator 1 of the host to be significantly down regulated during the entire infection period. Our data support the hypothesis that apoptosis of honey bee cells was suppressed during infection. We further analyzed genome-wide genetic diversity of this parasite by comparing samples collected from the same site in 2007 and 2013. The number of SNP positions per gene and the proportion of non-synonymous substitutions per gene were significantly reduced over this time period, suggesting purifying selection on the parasite genome and supporting the hypothesis that a subset of N. ceranae strains might be dominating infection. PMID:26840596
Host-Parasite Interactions and Purifying Selection in a Microsporidian Parasite of Honey Bees.

PubMed

Huang, Qiang; Chen, Yan Ping; Wang, Rui Wu; Cheng, Shang; Evans, Jay D

2016-01-01

To clarify the mechanisms of Nosema ceranae parasitism, we deep-sequenced both honey bee host and parasite mRNAs throughout a complete 6-day infection cycle. By time-series analysis, 1122 parasite genes were significantly differently expressed during the reproduction cycle, clustering into 4 expression patterns. We found reactive mitochondrial oxygen species modulator 1 of the host to be significantly down regulated during the entire infection period. Our data support the hypothesis that apoptosis of honey bee cells was suppressed during infection. We further analyzed genome-wide genetic diversity of this parasite by comparing samples collected from the same site in 2007 and 2013. The number of SNP positions per gene and the proportion of non-synonymous substitutions per gene were significantly reduced over this time period, suggesting purifying selection on the parasite genome and supporting the hypothesis that a subset of N. ceranae strains might be dominating infection.
Role of laccase from Coriolus versicolor MTCC-138 in selective oxidation of aromatic methyl group.

PubMed

Chaurasia, Pankaj Kumar; Singh, Sunil Kumar; Bharati, Shashi Lata

2014-01-01

Now a day, laccases are the most promising enzymes in the area of biotechnology and synthesis. One of the best applications of laccases is the selective oxidation of aromatic methyl group to aldehyde group. Such transformations are valuable because it is difficult to stop the reaction at aldehyde stage. Chemical methods used for such biotransformations areexpensive and give poor yields. But, the laccase-catalyzed biotransformations of such type are non-expensive and yield is excellent. Authors have used crude laccase obtained from the liquid culture growth medium of fungal strain Coriolus versicolor MTCC-138 for the biotransformations of toluene, 3-nitrotoluene, and 4-chlorotoluene to benzaldehyde, 3-nitrobenzaldehyde, and 4-chlorobenzaldehyde, respectively, instead of purified laccase because purification process requires much time and cost. This communication reports that crude laccase can also be used in the place of purified laccase as effective biocatalyst.

Entanglement of purification in free scalar field theories

NASA Astrophysics Data System (ADS)

Bhattacharyya, Arpan; Takayanagi, Tadashi; Umemoto, Koji

2018-04-01

We compute the entanglement of purification (EoP) in a 2d free scalar field theory with various masses. This quantity measures correlations between two subsystems and is reduced to the entanglement entropy when the total system is pure. We obtain explicit numerical values by assuming minimal gaussian wave functionals for the purified states. We find that when the distance between the subsystems is large, the EoP behaves like the mutual information. However, when the distance is small, the EoP shows a characteristic behavior which qualitatively agrees with the conjectured holographic computation and which is different from that of the mutual information. We also study behaviors of mutual information in purified spaces and violations of monogamy/strong superadditivity.
Effects of Simulated Hypogravity and Diet on Estrous Cycling in Rats

NASA Technical Reports Server (NTRS)

Tou, Janet C.; Grindeland, Richard E.; Baer, Lisa A.; Wade, Charles E.

2003-01-01

Environmental factors can disrupt ovulatory cycles. The study objective was to determine the effect of diet and simulated hypogravity on rat estrous cycles. Age 50 d Sprague-Dawley rats were randomly assigned to he fed either a purified or chow diet. Only normal cycling rats were used. Experimental rats (n=9-10/group) were kept as ambulatory controls (AC) or subjected to 40 d simulated hypogravity using a disuse atrophy hindlimb suspension (HLS) model. There was no effect on estrous cycles of AC fed either diet. At day 18, HLS rats fed either diet, had lengthened estrous cycles due to prolonged diestrus. HLS rats fed purified diet also had reduced time in estrus. Plasma estradiol was reduced in HLS rats fed purified diet but there was no effect on progesterone. This may have occurred because blood was collected from rats in estrus. Urinary progesterone collected during initial HLS was elevated in rats fed purified diet. In AC, corticosterone was elevated in chow vs purified diet fed rats. Differences were particularly striking following the application of a stressor with HLS/chow-fed rats displaying an enhanced stress response. Results emphasize the importance of diet selection when measuring endocrine-sensitive endpoints. HLS is a useful model for investigating the effects of environment on reproduction and providing insight about the impact extreme environment such as spaceflight on female reproductive health.
Disease-associated mitochondrial mutations and the evolution of primate mitogenomes

PubMed Central

Tavares, William Corrêa

2017-01-01

Several human diseases have been associated with mutations in mitochondrial genes comprising a set of confirmed and reported mutations according to the MITOMAP database. An analysis of complete mitogenomes across 139 primate species showed that most confirmed disease-associated mutations occurred in aligned codon positions and gene regions under strong purifying selection resulting in a strong evolutionary conservation. Only two confirmed variants (7.1%), coding for the same amino acids accounting for severe human diseases, were identified without apparent pathogenicity in non-human primates, like the closely related Bornean orangutan. Conversely, reported disease-associated mutations were not especially concentrated in conserved codon positions, and a large fraction of them occurred in highly variable ones. Additionally, 88 (45.8%) of reported mutations showed similar variants in several non-human primates and some of them have been present in extinct species of the genus Homo. Considering that recurrent mutations leading to persistent variants throughout the evolutionary diversification of primates are less likely to be severely damaging to fitness, we suggest that these 88 mutations are less likely to be pathogenic. Conversely, 69 (35.9%) of reported disease-associated mutations occurred in extremely conserved aligned codon positions which makes them more likely to damage the primate mitochondrial physiology. PMID:28510580
Mechanism of inhibition of adenovirus DNA replication by the acyclic nucleoside triphosphate analogue (S)-HPMPApp: influence of the adenovirus DNA binding protein.

PubMed Central

Mul, Y M; van Miltenburg, R T; De Clercq, E; van der Vliet, P C

1989-01-01

The acyclic adenosine analogue (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine [S]-HPMPA) is a potent and selective inhibitor of adenovirus (Ad) replication in cell culture. We studied the mechanism of inhibition using a reconstituted in vitro DNA replication system. The diphosphoryl derivative (S)-HPMPApp, but not (S)-HPMPA, inhibited the DNA replication of origin containing fragments strongly. The inhibitory effect was exerted at the level of elongation, while initiation was resistant to the drug. Remarkably, the elongation of short strands was only slightly impaired, while inhibition was maximal upon synthesis of long DNA fragments. (S)-HPMPApp appeared to be competitive with dATP, suggesting that the Ad DNA polymerase is the prime target for the drug. We purified the Ad DNA polymerase in complex to the precursor terminal protein to homogeneity from cells infected with overproducing recombinant vaccinia viruses. Employing gapped DNA or poly(dT).oligo(dA) templates, only a weak inhibition was observed. However, inhibition was strongly enhanced in the presence of the adenovirus DNA binding protein (DBP). We interpret this to mean that the increased processivity of the polymerization reaction in the presence of DBP leads to increased drug sensitivity. Images PMID:2587248
Selection and characterization of a mutant of feline immunodeficiency virus resistant to 2',3'-dideoxycytidine.

PubMed Central

Medlin, H K; Zhu, Y Q; Remington, K M; Phillips, T R; North, T W

1996-01-01

We have selected and plaque purified a mutant of feline immunodeficiency virus (FIV) that is resistant to 2',3'-dideoxycytidine (ddC). This mutant was selected in cultured cells in the continuous presence of 25 microM ddC. The mutant, designated DCR-5c, was fourfold resistant to ddC, threefold resistant to 2',3'-dideoxyinosine, and more than fourfold resistant to phosphonoformic acid. DCR-5c displayed little or no resistance to (-)-beta-2',3'-dideoxy-3'-thiacytidine, 3'-azido-3'-deoxythymidine, or 9-(2-phosphonylmethoxyethyl) adenine. Reverse transcriptase purified from DCR-5c was less susceptible to inhibition by ddCTP, phosphonoformic acid, ddATP, or azido-dTTP than the wild-type FIV reverse transcriptase. Sequence analysis of DCR-5c revealed a single base change (G to C at nucleotide 2342) in the reverse transcriptase-encoding region of FIV. This mutation results in substitution of His for Asp at codon 3 of FIV reverse transcriptase. The role of this mutation in ddC resistance was confirmed by site-directed mutagenesis. PMID:8849258
Spectroscopy reveals that ethyl esters interact with proteins in wine.

PubMed

Di Gaspero, Mattia; Ruzza, Paolo; Hussain, Rohanah; Vincenzi, Simone; Biondi, Barbara; Gazzola, Diana; Siligardi, Giuliano; Curioni, Andrea

2017-02-15

Impairment of wine aroma after vinification is frequently associated to bentonite treatments and this can be the result of protein removal, as recently demonstrated for ethyl esters. To evaluate the existence of an interaction between wine proteins and ethyl esters, the effects induced by these fermentative aroma compounds on the secondary structure and stability of VVTL1, a Thaumatin-like protein purified from wine, was analyzed by Synchrotron Radiation Circular Dichroism (SRCD) spectroscopy. The secondary structure of wine VVTL1 was not strongly affected by the presence of selected ethyl esters. In contrast, VVTL1 stability was slightly increased by the addition of ethyl-octanoate, -decanoate and -dodecanoate, but decreased by ethyl-hexanoate. This indicates the existence of an interaction between VVTL1 and at least some aroma compounds produced during fermentation. The data suggest that proteins removal from wine by bentonite can result in indirect removal of at least some aroma compounds associated with them. Copyright © 2016 Elsevier Ltd. All rights reserved.
Comparative study of the antioxidant activities of some lipase-catalyzed alkyl dihydrocaffeates synthesized in ionic liquid.

PubMed

Gholivand, Somayeh; Lasekan, Ola; Tan, Chin Ping; Abas, Faridah; Wei, Leong Sze

2017-06-01

The solubility limitations of phenolic acids in many lipidic environments are now greatly improved by their enzymatic esterification in ionic liquids (ILs). Herein, four different ILs were tested for the esterification of dihydrocaffeic acid with hexanol and the best IL was selected for the synthesis of four other n-alkyl esters with different chain-lengths. The effect of alkyl chain length on the anti-oxidative properties of the resulted purified esters was investigated using β-carotene bleaching (BCB) and free radical scavenging method DPPH and compared with butylated hydroxytoluene (BHT) as reference compound. All four esters (methyl, hexyl, dodecyl and octadecyl dihydrocaffeates) exhibited relatively strong radical scavenging abilities. The scavenging activity of the test compounds was in the following order: methyl ester>hexyl ester⩾dodecyl ester>octadecyl ester>BHT while the order for the BCB anti-oxidative activity was; BHT>octadecyl ester>dodecyl ester>hexyl ester>methyl ester. Copyright © 2016 Elsevier Ltd. All rights reserved.
Extensive mitochondrial heteroplasmy in the neotropical ants of the Ectatomma ruidum complex (Formicidae: Ectatomminae).

PubMed

Meza-Lázaro, Rubi N; Poteaux, Chantal; Bayona-Vásquez, Natalia J; Branstetter, Michael G; Zaldívar-Riverón, Alejandro

2018-01-31

We assembled mitogenomes from 21 ant workers assigned to four morphospecies (E. ruidum spp. 1-4) and putative hybrids of the Ectatomma ruidum complex (E. ruidum spp. 2x3), and to E. tuberculatum using NGS data. Mitogenomes from specimens of E. ruidum spp. 3, 4 and 2 × 3 had a high proportion of polymorphic sites. We investigated whether polymorphisms in mitogenomes are due to nuclear mt paralogues (numts) or due to the presence of more than one mitogenome within an individual (heteroplasmy). We did not find loss of function signals in polymorphic protein-coding genes, and observed strong evidence for purifying selection in two haplotype-phased genes, which indicate the presence of two functional mitochondrial genomes coexisting within individuals instead of numts. Heteroplasmy due to hybrid paternal leakage is not supported by phylogenetic analyses. Our results reveal the presence of a fast-evolving secondary mitochondrial lineage of uncertain origin in the E. ruidum complex.
Molecular evolution of the HD-ZIP I gene family in legume genomes.

PubMed

Li, Zhen; Jiang, Haiyang; Zhou, Lingyan; Deng, Lin; Lin, Yongxiang; Peng, Xiaojian; Yan, Hanwei; Cheng, Beijiu

2014-01-01

Homeodomain leucine zipper I (HD-ZIP I) genes were used to increase the plasticity of plants by mediating external signals and regulating growth in response to environmental conditions. The way genomic histories drove the evolution of the HD-ZIP I family in legume species was described; HD-ZIP I genes were searched in Lotus japonicus, Medicago truncatula, Cajanus cajan and Phaseolus vulgaris, and then divided into five clades through phylogenetic analysis. Microsynteny analysis was made based on genomic segments containing the HD-ZIP I genes. Some pairs turned out to conform with syntenic genome regions, while others corresponded to those that were inverted, expanded, or contracted after the divergence of legumes. Besides, we dated their duplications by Ks analysis and demonstrated that all the blocks were formed after the monocot-dicot split; we observed Ka/Ks ratios representing strong purifying selections in the four legume species which might have been followed by gene loss and rearrangement. © 2014 Elsevier B.V. All rights reserved.
Method for refining contaminated iridium

DOEpatents

Heshmatpour, B.; Heestand, R.L.

1982-08-31

Contaminated iridium is refined by alloying it with an alloying agent selected from the group consisting of manganese and an alloy of manganese and copper, and then dissolving the alloying agent from the formed alloy to provide a purified iridium powder.
A cherry protein and its gene, abundantly expressed in ripening fruit, have been identified as thaumatin-like.

PubMed

Fils-Lycaon, B R; Wiersma, P A; Eastwell, K C; Sautiere, P

1996-05-01

A 29-kD polypeptide is the most abundant soluble protein in ripe cherry fruit (Prunus avium L); accumulation begins at the onset of ripening as the fruit turns from yellow to red. This protein was extracted from ripe cherries and purified by size-exclusion and ion-exchange chromatography. Antibodies to the purified protein were used to screen a cDNA library from ripe cherries. Numerous recombinant plaques reacted positively with the antibodies; the DNA sequence of representative clones encoded a polypeptide of 245 amino acid residues. A signal peptide was indicated, and the predicted mature protein corresponded to the purified protein in size (23.3 kD, by mass spectrometry) and isoelectric point (4.2). A search of known protein sequences revealed a strong similarity between this polypeptide and the thaumatin family of pathogenesis-related proteins. The cherry thaumatin-like protein does not have a sweet taste, and no antifungal activity was seen in preliminary assays. Expression of the protein appears to be regulated at the gene level, with mRNA levels at their highest in the ripe fruit.
A cherry protein and its gene, abundantly expressed in ripening fruit, have been identified as thaumatin-like.

PubMed Central

Fils-Lycaon, B R; Wiersma, P A; Eastwell, K C; Sautiere, P

1996-01-01

A 29-kD polypeptide is the most abundant soluble protein in ripe cherry fruit (Prunus avium L); accumulation begins at the onset of ripening as the fruit turns from yellow to red. This protein was extracted from ripe cherries and purified by size-exclusion and ion-exchange chromatography. Antibodies to the purified protein were used to screen a cDNA library from ripe cherries. Numerous recombinant plaques reacted positively with the antibodies; the DNA sequence of representative clones encoded a polypeptide of 245 amino acid residues. A signal peptide was indicated, and the predicted mature protein corresponded to the purified protein in size (23.3 kD, by mass spectrometry) and isoelectric point (4.2). A search of known protein sequences revealed a strong similarity between this polypeptide and the thaumatin family of pathogenesis-related proteins. The cherry thaumatin-like protein does not have a sweet taste, and no antifungal activity was seen in preliminary assays. Expression of the protein appears to be regulated at the gene level, with mRNA levels at their highest in the ripe fruit. PMID:8685266
The HSV-1 tegument protein pUL46 associates with cellular membranes and viral capsids

DOE Office of Scientific and Technical Information (OSTI.GOV)

Murphy, Michael A.; Bucks, Michelle A.; O'Regan, Kevin J.

2008-07-05

The molecular mechanisms responsible for the addition of tegument proteins into nascent herpesvirus particles are poorly understood. To better understand the tegumentation process of herpes simplex virus type 1 (HSV-1) virions, we initiated studies that showed the tegument protein pUL46 (VP11/12) has a similar cellular localization to the membrane-associated tegument protein VP22. Using membrane flotation analysis we found that pUL46 associates with membranes in both the presence and absence of other HSV-1 proteins. However, when purified virions were stripped of their envelope, the majority of pUL46 was found to associate with the capsid fraction. This strong affinity of pUL46 formore » capsids was confirmed by an in vitro capsid pull-down assay in which purified pUL46-GST was able to interact specifically with capsids purified from the nuclear fraction of HSV-1 infected cells. These results suggest that pUL46 displays a dynamic interaction between cellular membranes and capsids.« less
Simplified in vitro refolding and purification of recombinant human granulocyte colony stimulating factor using protein folding cation exchange chromatography.

PubMed

Vemula, Sandeep; Dedaniya, Akshay; Thunuguntla, Rahul; Mallu, Maheswara Reddy; Parupudi, Pavani; Ronda, Srinivasa Reddy

2015-01-30

Protein folding-strong cation exchange chromatography (PF-SCX) has been employed for efficient refolding with simultaneous purification of recombinant human granulocyte colony stimulating factor (rhG-CSF). To acquire a soluble form of renatured and purified rhG-CSF, various chromatographic conditions, including the mobile phase composition and pH was evaluated. Additionally, the effects of additives such as urea, amino acids, polyols, sugars, oxidizing agents and their amalgamations were also investigated. Under the optimal conditions, rhG-CSF was efficaciously solubilized, refolded and simultaneously purified by SCX in a single step. The experimental results using ribose (2.0M) and arginine (0.6M) combination were found to be satisfactory with mass yield, purity and specific activity of 71%, ≥99% and 2.6×10(8)IU/mg respectively. Through this investigation, we concluded that the SCX refolding method was more efficient than conventional methods which has immense potential for the large-scale production of purified rhG-CSF. Copyright © 2014 Elsevier B.V. All rights reserved.
Anti-Plasmodial Polyvalent Interactions in Artemisia annua L. Aqueous Extract – Possible Synergistic and Resistance Mechanisms

PubMed Central

Suberu, John O.; Gorka, Alexander P.; Jacobs, Lauren; Roepe, Paul D.; Sullivan, Neil

2013-01-01

Artemisia annua hot water infusion (tea) has been used in in vitro experiments against P. falciparum malaria parasites to test potency relative to equivalent pure artemisinin. High performance liquid chromatography (HPLC) and mass spectrometric analyses were employed to determine the metabolite profile of tea including the concentrations of artemisinin (47.5±0.8 mg L-1), dihydroartemisinic acid (70.0±0.3 mg L-1), arteannuin B (1.3±0.0 mg L-1), isovitexin (105.0±7.2 mg L-1) and a range of polyphenolic acids. The tea extract, purified compounds from the extract, and the combination of artemisinin with the purified compounds were tested against chloroquine sensitive and chloroquine resistant strains of P. falciparum using the DNA-intercalative SYBR Green I assay. The results of these in vitro tests and of isobologram analyses of combination effects showed mild to strong antagonistic interactions between artemisinin and the compounds (9-epi-artemisinin and artemisitene) extracted from A. annua with significant (IC50 <1 μM) anti-plasmodial activities for the combination range evaluated. Mono-caffeoylquinic acids, tri-caffeoylquinic acid, artemisinic acid and arteannuin B showed additive interaction while rosmarinic acid showed synergistic interaction with artemisinin in the chloroquine sensitive strain at a combination ratio of 1:3 (artemisinin to purified compound). In the chloroquine resistant parasite, using the same ratio, these compounds strongly antagonised artemisinin anti-plasmodial activity with the exception of arteannuin B, which was synergistic. This result would suggest a mechanism targeting parasite resistance defenses for arteannuin B’s potentiation of artemisinin. PMID:24244716
Purification and Characterization of Two Distinct NAD(P)H Dehydrogenases from Onion (Allium cepa L.) Root Plasma Membrane.

PubMed Central

Serrano, A.; Cordoba, F.; Gonzalez-Reyes, J. A.; Navas, P.; Villalba, J. M.

1994-01-01

Highly purified plasma membrane fractions were obtained from onion (Allium cepa L.) roots and used as a source for purification of redox proteins. Plasma membranes solubilized with Triton X-100 contained two distinct polypeptides showing NAD(P)H-dependent dehydrogenase activities. Dehydrogenase I was purified by gel filtration in Sephacryl S-300 HR, ion-exchange chromatography in DEAE-Sepharose CL-6B, and dye-ligand affinity chromatography in Blue-Sepharose CL-6B after biospecific elution with NADH. Dehydrogenase I consisted of a single polypeptide of about 27 kD and an isoelectric point of about 6. Dehydrogenase II was purified from the DEAE-unbound fraction by chromatography in Blue-Sepharose CL-6B and affinity elution with NADH. Dehydrogenase II consisted of a single polypeptide of about 31 kD and an isoelectric point of about 8. Purified dehydrogenase I oxidized both NADPH and NADH, although higher rates of electron transfer were obtained with NADPH. Maximal activity was achieved with NADPH as donor and juglone or coenzyme Q as acceptor. Dehydrogenase II was specific for NADH and exhibited maximal activity with ferricyanide. Optimal pH for both dehydrogenases was about 6. Dehydrogenase I was moderately inhibited by dicumarol, thenoyltrifluoroacetone, and the thiol reagent N-ethyl-maleimide. A strong inhibition of dehydrogenase II was obtained with dicumarol, thenoyltrifluoroacetone, and the thiol reagent p-hydroxymercuribenzoate. PMID:12232306
An optimal thermal evaporation synthesis of c-axis oriented ZnO nanowires with excellent UV sensing and emission characteristics

DOE Office of Scientific and Technical Information (OSTI.GOV)

Saha, Tridib, E-mail: tridib.saha@monash.edu; Achath Mohanan, Ajay, E-mail: ajay.mohanan@monash.edu; Swamy, Varghese, E-mail: varghese.swamy@monash.edu

Highlights: • c-Axis alignment of ZnO nanowires was optimized using self-seeding thermal evaporation method. • Influence of purified air on the morphology and optoelectronic properties were studied. • Nanowires grown under optimal conditions exhibit strong UV emission peak in PL spectrum. • Optimized growth condition establish nanowires of excellent UV sensing characteristics - Abstract: Well-aligned (c-axis oriented) ZnO nanowire arrays were successfully synthesized on Si (1 0 0) substrates through an optimized self-seeding thermal evaporation method. An open-ended chemical vapor deposition (CVD) setup was used in the experiment, with argon and purified air as reaction gases. Epitaxial growth of c-axismore » oriented ZnO nanowires was observed for 5 sccm flow rate of purified air, whereas Zn/Zn suboxide layers and multiple polycrystalline layers of ZnO were obtained for absence and excess of purified air, respectively. Ultraviolet (UV) sensing and emission properties of the as-grown ZnO nanostructures were investigated through the current–voltage (I–V) characteristics of the nanowires under UV (λ = 365 nm) illumination of 8 mW/cm{sup 2} and using photoluminescence spectra. Nanowires grown under optimum flow of air emitted four times higher intensity of 380 nm UV light as well as exhibited 34 times higher UV radiation sensitivity compared to that of other nanostructures synthesized in this study.« less
Synthesis and characterization of nano-sized CaCO3 in purified diet

NASA Astrophysics Data System (ADS)

Mulyaningsih, N. N.; Tresnasari, D. R.; Ramahwati, M. R.; Juwono, A. L.; Soejoko, D. S.; Astuti, D. A.

2017-07-01

The growth and development of animals depend strongly on the balanced nutrition in the diet. This research aims is to characterize the weight variations of nano-sized calcium carbonate (CaCO3) in purified diet that to be fed to animal model of rat. The nano-sized CaCO3 was prepared by milling the calcium carbonate particles for 20 hours at a rotation speed of 1000 rpm and resulting particle size in a range of 2-50 nm. Nano-sized CaCO3 added to purified diet to the four formulas that were identified as normal diet (N), deficiency calcium (DC), rich in calcium (RC), and poor calcium (PC) with containing in nano-sized CaCO3 much as 0.50 %, 0.00 %, 0.75 % and 0.25 % respectively. The nutritional content of the purified diet was proximate analyzed, it resulted as followed moisture, ash, fat, protein, crude fiber. The quantities of chemical element were analyzed by atomic absorption spectrometry (AAS), it resulted iron, magnesium, potassium and calcium. The results showed that N diet (Ca: 16,914.29 ppm) were suggested for healthy rats and RC diet (Ca: 33,696.13 ppm) for conditioned osteoporosis rats. The crystalline phases of the samples that were examined by X-ray diffraction showed that crystalline phase increased with the increasing concentration of CaCO3.
A novel reusable nanocomposite for complete removal of dyes, heavy metals and microbial load from water based on nanocellulose and silver nano-embedded pebbles.

PubMed

Suman; Kardam, Abhishek; Gera, Meeta; Jain, V K

2015-01-01

The present work proposed a nanocellulose (NC)-silver nanoparticles (AgNPs) embedded pebbles-based composite material as a novel reusable cost-effective water purification device for complete removal of dyes, heavy metals and microbes. NC was prepared using acid hydrolysis of cellulose. The AgNPs were generated in situ using glucose and embedded within the porous concrete pebbles by the technique of inter-diffusion of ion, providing a very strong binding of nanoparticles within the porous pebbles and thus preventing any nanomaterials leaching. Fabrication of a continual running water purifier was achieved by making different layering of NC and Ag nano-embedded pebbles in a glass column. The water purifier exhibited not only excellent dye and heavy metal adsorption capacity, but also long-term antibacterial activity against pathogenic and non-pathogenic bacterial strains. The adsorption mainly occurred through electrostatic interaction and pore diffusion also contributed to the process. The bed column purifier has shown 99.48% Pb(II) and 98.30% Cr(III) removal efficiency along with 99% decontamination of microbial load at an optimum working pH of 6.0. The high adsorption capacity and reusability, with complete removal of dyes, heavy metals and Escherichia coli from the simulated contaminated water of composite material, will provide new opportunities to develop a cost-effective and eco-friendly water purifier for commercial application.
Extreme primary and secondary protein structure variability in the chimeric male-transmitted cytochrome c oxidase subunit II protein in freshwater mussels: Evidence for an elevated amino acid substitution rate in the face of domain-specific purifying selection

PubMed Central

2008-01-01

Background Freshwater unionoidean bivalves, and species representing two marine bivalve orders (Mytiloida and Veneroida), exhibit a mode of mtDNA inheritance involving distinct maternal (F) and paternal (M) transmission routes concomitant with highly divergent gender-associated mtDNA genomes. Additionally, male unionoidean bivalves have a ~550 bp 3' coding extension to the cox2 gene (Mcox2e), that is apparently absent from all other metazoan taxa. Results Our molecular sequence analyses of MCOX2e indicate that both the primary and secondary structures of the MCOX2e region are evolving much faster than other regions of the F and M COX2-COX1 gene junction. The near N-terminus ~2/3 of the MCOX2e region contains an interspecifically variable number of predicted transmembrane helices (TMH) and interhelical loops (IHL) whereas the C-terminus ~1/3 is relatively conserved and hydrophilic while containing conserved functional motifs. MCOX2e displays an overall pattern of purifying selection that leads to the preservation of TMH/IHL and C-terminus tail sub-regions. However, 14 amino acid positions in the MCOX2e TMH/IHL sub-region might be targeted by diversifying selection, each representing a site where there exists interspecific variation for the constituent amino acids residing in a TMH or IHL. Conclusion Our results indicate that Mcox2e is unique to unionoidean bivalves, likely the result of a single insertion event that took place over 65 MYA and that MCOX2e is functional. The predicted TMH number, length and position variability likely stems from substitution-based processes rather than the typically implicated insertion/deletion events. MCOX2e has relatively high rates of primary and secondary structure evolution, with some amino acid residues potentially subjected to site-specific positive selection, yet an overall pattern of purifying selection leading to the preservation of the TMH/IHL and hydrophilic C-terminus tail subregions. The more conserved C-terminus tail (relative to the TMH/IHL sub-region of MCOX2e) is likely biologically active because it contains functional motifs. The rapid evolution of primary and secondary structure in MCOX2e, combined with the action of both positive and purifying selection, provide supporting evidence for the hypothesis that MCOX2e has a novel reproductive function within unionoidean bivalves. All tolled, our data indicate that unionoidean bivalve MCOX2 is the first reported chimeric animal mtDNA-encoded protein. PMID:18513440

The feasibility of an air purifier and secondhand smoke education intervention in homes of inner city pregnant women and infants living with a smoker.

PubMed

Rice, Jessica L; Brigham, Emily; Dineen, Rebecca; Muqueeth, Sadiya; O'Keefe, Gena; Regenold, Stephanie; Koehler, Kirsten; Rule, Ana; McCormack, Meredith; Hansel, Nadia N; Diette, Gregory B

2018-01-01

Secondhand smoke (SHS) and other air pollutants adversely affect the health of pregnant women and infants. A feasibility study aimed at reducing air pollution in homes of pregnant women or infants living with a smoker was completed. In collaboration with the Baltimore City Health Department, women ≥ 18 years of age and either pregnant nonsmokers, or post-partum (any smoking status) with an infant age 0-12 months were recruited. Homes had at least one smoker. Intervention included two air purifiers and secondhand smoke education. Outcomes included feasibility, change in fine particulate matter (PM 2.5 ), air nicotine, and salivary cotinine pre- and post-intervention. Fifty women were enrolled (mean age 27 years, 92% African American, 71% single, 94% Medicaid eligible, 34% reported smoking) and 86% completed the study. Of the 50 women, 32 had infants and 18 were pregnant at time of enrollment. Post- intervention, 70% of participants reported smokers were less likely to smoke indoors, and 77% had at least one air purifier turned on at the final visit. Participant satisfaction was high (91%) and 98% would recommend air purifiers. Indoor PM 2.5 was significantly decreased (P < 0.001). Salivary cotinine was significantly decreased for non-smoking women (P < 0.01) but not infants, and no significant change in air nicotine occurred (P = 0.6). Air purifiers with SHS education is a feasible intervention in homes of women and infants. These data demonstrate reduction in indoor PM 2.5 and salivary cotinine in non-smoking adults. Air purifiers are not an alternative for smoking cessation and a home/ car smoking ban. Smoking cessation should be strongly encouraged for all pregnant women, and nonsmoking mothers with infants should be counseled to completely avoid SHS exposure. This study provides support for a future intervention evaluating clinical endpoints. Copyright © 2017. Published by Elsevier Inc.
Bovine pancreatic trypsin inhibitor immobilized onto sepharose as a new strategy to purify a thermostable alkaline peptidase from cobia (Rachycentron canadum) processing waste.

PubMed

França, Renata Cristina da Penha; Assis, Caio Rodrigo Dias; Santos, Juliana Ferreira; Torquato, Ricardo José Soares; Tanaka, Aparecida Sadae; Hirata, Izaura Yoshico; Assis, Diego Magno; Juliano, Maria Aparecida; Cavalli, Ronaldo Olivera; Carvalho, Luiz Bezerra de; Bezerra, Ranilson Souza

2016-10-15

A thermostable alkaline peptidase was purified from the processing waste of cobia (Rachycentron canadum) using bovine pancreatic trypsin inhibitor (BPTI) immobilized onto Sepharose. The purified enzyme had an apparent molecular mass of 24kDa by both sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry. Its optimal temperature and pH were 50°C and 8.5, respectively. The enzyme was thermostable until 55°C and its activity was strongly inhibited by the classic trypsin inhibitors N-ρ-tosyl-l-lysine chloromethyl ketone (TLCK) and benzamidine. BPTI column allowed at least 15 assays without loss of efficacy. The purified enzyme was identified as a trypsin and the N-terminal amino acid sequence of this trypsin was IVGGYECTPHSQAHQVSLNSGYHFC, which was highly homologous to trypsin from cold water fish species. Using Nα-benzoyl-dl-arginine ρ-nitroanilide hydrochloride (BApNA) as substrate, the apparent km value of the purified trypsin was 0.38mM, kcat value was 3.14s(-1), and kcat/km was 8.26s(-1)mM(-1). The catalytic proficiency of the purified enzyme was 2.75×10(12)M(-1) showing higher affinity for the substrate at the transition state than other fish trypsin. The activation energy (AE) of the BApNA hydrolysis catalyzed by this enzyme was estimated to be 11.93kcalmol(-1) while the resulting rate enhancement of this reaction was found to be approximately in a range from 10(9) to 10(10)-fold evidencing its efficiency in comparison to other trypsin. This new purification strategy showed to be appropriate to obtain an alkaline peptidase from cobia processing waste with high purification degree. According with N-terminal homology and kinetic parameters, R. canadum trypsin may gathers desirable properties of psychrophilic and thermostable enzymes. Copyright © 2016 Elsevier B.V. All rights reserved.
Purification and characterization of an alkaline protease from the marine yeast Aureobasidium pullulans for bioactive peptide production from different sources.

PubMed

Ma, Chunling; Ni, Xiumei; Chi, Zhenming; Ma, Liyan; Gao, Lingmei

2007-01-01

The extracellular alkaline protease in the supernatant of cell culture of the marine yeast Aureobasidium pullulans 10 was purified to homogeneity with a 2.1-fold increase in specific protease activity as compared to that in the supernatant by ammonium sulfate fractionation, gel filtration chromatography (Sephadex G-75), and anion-exchange chromatography (DEAE Sepharose Fast Flow). According to the sodium dodecyl sulfate-polyacrylamide gel electrophoresis data, the molecular mass of the purified enzyme was estimated to be 32.0 kDa. The optimal pH and temperature of the purified enzyme were 9.0 and 45 degrees C, respectively. The enzyme was activated by Cu(2+) (at a concentration of 1.0 mM) and Mn(2+) and inhibited by Hg(2+), Fe(2+), Fe(3+), Zn(2+), and Co(2+). The enzyme was strongly inhibited by phenylmethylsulfonyl fluoride, but weakly inhibited by EDTA, 1-10-phenanthroline, and iodoacetic acid. The K(m) and V(max) values of the purified enzyme for casein were 0.25 mg/ml and 0.0286 micromol/min/mg of protein, respectively. After digestion of shrimp protein, spirulina (Arthospira platensis) protein, proteins of marine yeast strains N3C (Yarrowia lipolytica) and YA03a (Hanseniaspora uvarum), milk protein, and casein with the purified alkaline protease, angiotensin I converting enzyme (ACE) inhibitory activities of the resulting peptides reached 85.3%, 12.1%, 29.8%, 22.8%, 14.1%, and 15.5%, respectively, while the antioxidant activities of these were 52.1%. 54.6%, 25.1%, 35%, 12.5%, and 24.2%, respectively, indicating that ACE inhibitory activity of the resulting peptides from the shrimp protein and antioxidant activity of those produced from the spirulina protein were the highest, respectively. These results suggest that the bioactive peptides produced by digestion of the shrimp protein with the purified alkaline protease have potential applications in the food and pharmaceutical industries.
Apparatus for purifying arsine, phosphine, ammonia, and inert gases to remove Lewis acid and oxidant impurities therefrom

DOEpatents

Tom, Glenn M.; Brown, Duncan W.

1991-01-08

An apparatus for purifying a gaseous mixture comprising arsine, phosphine, ammonia, and/or inert gases, to remove Lewis acid and/or oxidant impurities therefrom, comprising a vessel containing a bed of a scavenger, the scavenger including a support having associated therewith an anion which is effective to remove such impurities, such anion being selected from one or more members of the group consisting of: (i) carbanions whose corresponding protonated compounds have a pK.sub.a value of from about 22 to about 36; and (ii) anions formed by reaction of such carbanions with the primary component of the mixture.
Ozone removal capability of a welding fume respirator containing activated charcoal.

PubMed

Johnston, A R; Dyrud, J F; Shih, Y T

1989-09-01

Development of air purifying respirators for protection against ozone has been slowed by concerns about oxidation of charcoal and other available sorbents. The suitability of a charcoal sorbent for low concentrations of ozone was evaluated as a part of the development of a half-mask air purifying respirator designed for welding fumes and ozone. Testing of the respirator confirmed that charcoal can be a suitable sorbent for low levels of ozone. Where the respirator is properly selected, fit tested, and worn, respirator use against welding fumes and ozone at concentrations not exceeding 10 times the permissible exposure limit had been recommended.
Selection of Human Antibody Fragments which Bind Novel Breast Tumor Antigens

DTIC Science & Technology

1996-09-01

OmniGene cycler. The products were gel C6.5 must occur rarely in the repertoire, since none purified, isolated from the gel using DEAE membranes, of 92...C6VHCDR3A, C6VHCDR3B, C6VHCDR3C, and minute) using a Hybaid OmniGene cycler. To introduce C6VHCDR3D. a Notl restriction site at the 3’ end of the scFv gene...34C for 20 30 sec, 55 *C for 30 sec and 72°C for 30 sec) using a Hybaid OmniGene cycler. The products were gel purified, isolated from the gel using
Technical adequacy of bisulfite sequencing and pyrosequencing for detection of mitochondrial DNA methylation: Sources and avoidance of false-positive detection.

PubMed

Owa, Chie; Poulin, Matthew; Yan, Liying; Shioda, Toshi

2018-01-01

The existence of cytosine methylation in mammalian mitochondrial DNA (mtDNA) is a controversial subject. Because detection of DNA methylation depends on resistance of 5'-modified cytosines to bisulfite-catalyzed conversion to uracil, examined parameters that affect technical adequacy of mtDNA methylation analysis. Negative control amplicons (NCAs) devoid of cytosine methylation were amplified to cover the entire human or mouse mtDNA by long-range PCR. When the pyrosequencing template amplicons were gel-purified after bisulfite conversion, bisulfite pyrosequencing of NCAs did not detect significant levels of bisulfite-resistant cytosines (brCs) at ND1 (7 CpG sites) or CYTB (8 CpG sites) genes (CI95 = 0%-0.94%); without gel-purification, significant false-positive brCs were detected from NCAs (CI95 = 4.2%-6.8%). Bisulfite pyrosequencing of highly purified, linearized mtDNA isolated from human iPS cells or mouse liver detected significant brCs (~30%) in human ND1 gene when the sequencing primer was not selective in bisulfite-converted and unconverted templates. However, repeated experiments using a sequencing primer selective in bisulfite-converted templates almost completely (< 0.8%) suppressed brC detection, supporting the false-positive nature of brCs detected using the non-selective primer. Bisulfite-seq deep sequencing of linearized, gel-purified human mtDNA detected 9.4%-14.8% brCs for 9 CpG sites in ND1 gene. However, because all these brCs were associated with adjacent non-CpG brCs showing the same degrees of bisulfite resistance, DNA methylation in this mtDNA-encoded gene was not confirmed. Without linearization, data generated by bisulfite pyrosequencing or deep sequencing of purified mtDNA templates did not pass the quality control criteria. Shotgun bisulfite sequencing of human mtDNA detected extremely low levels of CpG methylation (<0.65%) over non-CpG methylation (<0.55%). Taken together, our study demonstrates that adequacy of mtDNA methylation analysis using methods dependent on bisulfite conversion needs to be established for each experiment, taking effects of incomplete bisulfite conversion and template impurity or topology into consideration.
High-level expression and efficient purification of bioactive swollenin in Aspergillus oryzae.

PubMed

Wang, Meihua; Cai, Jin; Huang, Lei; Lv, Zhengbin; Zhang, Yaozhou; Xu, Zhinan

2010-11-01

The bioactivity of swollenin is beneficial to cellulose decomposition by cellulase despite the lack of hydrolytic activity itself. In order to improve the productivity of swollenin, the effects of culture conditions on the expression level in recombinant Aspergillus oryzae were investigated systematically. With regard to the bioactivity of swollenin, glycerin and peanut meal were the optimal carbon or nitrogen source, respectively. The highest level production of swollenin (50 mg L(-1)) was attained after 88 h cultivation with the initial pH of 5.6 in the culture medium. Then the soluble swollenin was effectively purified from the cultural supernatant by ammonium sulfate precipitation and cationic exchange chromatography with recovery yield of 53.2%. The purified swollenin was fully bioactive due to its strong synergistic activity with cellulose.
The Type 1 Homodimeric Reaction Center in Heliobacterium modesticaldum

DOE Office of Scientific and Technical Information (OSTI.GOV)

Golbeck, John

In this funding period, we (i) found that strong illumination of Heliobacterium modesticaldum cells results in saturation of the electron acceptor pool, leading to reduction of the acceptor side and the creation of a back-reacting state that gives rise to delayed fluorescence; (ii) noted that when the FX cluster is reduced in purified reaction centers, no electron transfer occurs beyond A0, even though a quinone is present; (iii) observed by photochemically induced dynamic nuclear polarization (photo-CIDNP) studies of whole cells of Heliobacterium mobilis that primary charge separation is retained even after conversion of the majority of BChl g to Chlmore » aF. ; and (iv) purified a homogeneous preparation of reaction center cores, which led to promising crystallization trials to obtain a three-dimensional structure.« less
[Expression, purification and antibody preparation of recombinat SARS-CoV X5 protein].

PubMed

Wang, Li-Na; Kong, Jian-Qiang; Zhu, Ping; Du, Guan-Hua; Wang, Wei; Cheng, Ke-Di

2008-11-01

X5 protein is one of the putative unknown proteins of SARS-CoV. The recombinant protein has been successfully expressed in E. coli in the form of insoluble inclusion body. The inclusion body was dissolved in high concentration of urea. Affinity Chromatography was preformed to purify the denatured protein, and then the product was refolded in a series of gradient solutions of urea. The purified protein was obtained with the purity of > 95% and the yield of 93.3 mg x L(-1). Polyclonal antibody of this protein was obtained, and Western blotting assay indicated that the X5 protein has the strong property of antigen. Sixty-eight percent of the recombinant protein sequence was confirmed by LC-ESI-MS/MS analysis.
Kraft Pulp Biobleaching and Mediated Oxidation of a Nonphenolic Substrate by Laccase from Streptomyces cyaneus CECT 3335

PubMed Central

Arias, M. Enriqueta; Arenas, María; Rodríguez, Juana; Soliveri, Juan; Ball, Andrew S.; Hernández, Manuel

2003-01-01

A new laccase (EC 1.10.3.2) produced by Streptomyces cyaneus CECT 3335 in liquid media containing soya flour (20 g per liter) was purified to homogeneity. The physicochemical, catalytic, and spectral characteristics of this enzyme, as well as its suitability for biobleaching of eucalyptus kraft pulps, were assessed. The purified laccase had a molecular mass of 75 kDa and an isoelectric point of 5.6, and its optimal pH and temperature were 4.5 and 70°C, respectively. The activity was strongly enhanced in the presence of Cu2+, Mn2+, and Mg2+ and was completely inhibited by EDTA and sodium azide. The purified laccase exhibited high levels of activity against 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) and 2,6-dimethoxyphenol and no activity against tyrosine. The UV-visible spectrum of the purified laccase was the typical spectrum of the blue laccases, with an absorption peak at 600 nm and a shoulder around 330 to 340 nm. The ability of the purified laccase to oxidize a nonphenolic compound, such as veratryl alcohol, in the presence of ABTS opens up new possibilities for the use of bacterial laccases in the pulp and paper industry. We demonstrated that application of the laccase from S. cyaneus in the presence of ABTS to biobleaching of eucalyptus kraft pulps resulted in a significant decrease in the kappa number (2.3 U) and an important increase in the brightness (2.2%, as determined by the International Standard Organization test) of pulps, showing the suitability of laccases produced by streptomycetes for industrial purposes. PMID:12676669
Assessment of hemolytic activity, enzyme production and bacteriocin characterization of Bacillus subtilis LR1 isolated from the gastrointestinal tract of fish.

PubMed

Banerjee, Goutam; Nandi, Ankita; Ray, Arun Kumar

2017-01-01

In the present investigation, probiotic potential (antagonistic activity, enzyme production, hemolytic activity, biosafety, antibiotic sensitivity and bile tolerance level) of Bacillus subtilis LR1 was evaluated. Bacteriocin produced by the bacterial strain B. subtilis LR1 isolated from the gastrointestinal tract of Labeo rohita was purified and characterized. The molecular weight of the purified bacteriocin was ~50 kDa in 12 % Native PAGE and showed inhibitory activity against four fish pathogens such as Bacillus mycoides, Aeromonas salmonicida, Pseudomonas fluorescens and Aeromonas hydrophila. The purified bacteriocin was maximally active at temperature 40 °C and pH 7.0, while none of the tested surfactants affect the bacteriocin activity. Extracellular enzyme activity of the selected bacterial strain was also evaluated. Amylase activity was estimated to be highest (38.23 ± 1.15 µg of maltose liberated mg -1 protein ml -1 of culture filtrate) followed by cellulase and protease activity. The selected bacterium was sensitive to most of the antibiotics used in this experiment, can tolerate 0.25 % bile salt and non-hemolytic in nature. Finally, the efficiency of the proposed probiotic candidate was evaluated in in vivo condition. It was detected that the bacterial strain can effectively reduce bacterial pathogenicity in Indian major carps.
Development of a Novel Human scFv Against EGFR L2 Domain by Phage Display Technology.

PubMed

Rahbarnia, Leila; Farajnia, Safar; Babaei, Hossein; Majidi, Jafar; Veisi, Kamal; Khosroshahi, Shiva Ahdi; Tanomand, Asghar

2017-01-01

Epidermal growth factor receptor (EGFR) as a transmembrane tyrosine kinase receptor frequently overexpresses in tumors with epithelial origin. The L2 domain from extracellular part of EGFR is involved in ligand binding and the blockage of this domain prevents activation of related signaling pathways. This study was aimed to develop a novel human scFv against EGFR L2 domain as a promising target for cancer therapy. The L2 recombinant protein was purified and used for panning a human scFv phage library (Tomlinson I). In this study, a novel screening strategy was applied to select clones with high binding and enrichment of rare specific phage clones of the L2 protein. After five biopanning rounds several specific clones were isolated which among them one phage clone with high binding was purified for further analysis. The specific interaction of selected clone against target antigen was confirmed by ELISA and western blotting. Immunofluorescence staining showed that purified scFv binds to A431 cells surface, displaying EGFR surface receptor. In the present study, we isolated for the first time a novel human scFv against EGFR L2 domain. This study can be the groundwork for developing more effective diagnostic and therapeutic agents against EGFR overexpressing cancers using this novel human anti-L2 ScFv. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.
Rapid diversification of five Oryza AA genomes associated with rice adaptation.

PubMed

Zhang, Qun-Jie; Zhu, Ting; Xia, En-Hua; Shi, Chao; Liu, Yun-Long; Zhang, Yun; Liu, Yuan; Jiang, Wen-Kai; Zhao, You-Jie; Mao, Shu-Yan; Zhang, Li-Ping; Huang, Hui; Jiao, Jun-Ying; Xu, Ping-Zhen; Yao, Qiu-Yang; Zeng, Fan-Chun; Yang, Li-Li; Gao, Ju; Tao, Da-Yun; Wang, Yue-Ju; Bennetzen, Jeffrey L; Gao, Li-Zhi

2014-11-18

Comparative genomic analyses among closely related species can greatly enhance our understanding of plant gene and genome evolution. We report de novo-assembled AA-genome sequences for Oryza nivara, Oryza glaberrima, Oryza barthii, Oryza glumaepatula, and Oryza meridionalis. Our analyses reveal massive levels of genomic structural variation, including segmental duplication and rapid gene family turnover, with particularly high instability in defense-related genes. We show, on a genomic scale, how lineage-specific expansion or contraction of gene families has led to their morphological and reproductive diversification, thus enlightening the evolutionary process of speciation and adaptation. Despite strong purifying selective pressures on most Oryza genes, we documented a large number of positively selected genes, especially those genes involved in flower development, reproduction, and resistance-related processes. These diversifying genes are expected to have played key roles in adaptations to their ecological niches in Asia, South America, Africa and Australia. Extensive variation in noncoding RNA gene numbers, function enrichment, and rates of sequence divergence might also help account for the different genetic adaptations of these rice species. Collectively, these resources provide new opportunities for evolutionary genomics, numerous insights into recent speciation, a valuable database of functional variation for crop improvement, and tools for efficient conservation of wild rice germplasm.
Evolutionary interplay between structure, energy and epistasis in the coat protein of the ϕX174 phage family.

PubMed

Redondo, Rodrigo A F; de Vladar, Harold P; Włodarski, Tomasz; Bollback, Jonathan P

2017-01-01

Viral capsids are structurally constrained by interactions among the amino acids (AAs) of their constituent proteins. Therefore, epistasis is expected to evolve among physically interacting sites and to influence the rates of substitution. To study the evolution of epistasis, we focused on the major structural protein of the ϕX174 phage family by first reconstructing the ancestral protein sequences of 18 species using a Bayesian statistical framework. The inferred ancestral reconstruction differed at eight AAs, for a total of 256 possible ancestral haplotypes. For each ancestral haplotype and the extant species, we estimated, in silico, the distribution of free energies and epistasis of the capsid structure. We found that free energy has not significantly increased but epistasis has. We decomposed epistasis up to fifth order and found that higher-order epistasis sometimes compensates pairwise interactions making the free energy seem additive. The dN/dS ratio is low, suggesting strong purifying selection, and that structure is under stabilizing selection. We synthesized phages carrying ancestral haplotypes of the coat protein gene and measured their fitness experimentally. Our findings indicate that stabilizing mutations can have higher fitness, and that fitness optima do not necessarily coincide with energy minima. © 2017 The Authors.
Rapid diversification of five Oryza AA genomes associated with rice adaptation

PubMed Central

Zhang, Qun-Jie; Zhu, Ting; Xia, En-Hua; Shi, Chao; Liu, Yun-Long; Zhang, Yun; Liu, Yuan; Jiang, Wen-Kai; Zhao, You-Jie; Mao, Shu-Yan; Zhang, Li-Ping; Huang, Hui; Jiao, Jun-Ying; Xu, Ping-Zhen; Yao, Qiu-Yang; Zeng, Fan-Chun; Yang, Li-Li; Gao, Ju; Tao, Da-Yun; Wang, Yue-Ju; Bennetzen, Jeffrey L.; Gao, Li-Zhi

2014-01-01

Comparative genomic analyses among closely related species can greatly enhance our understanding of plant gene and genome evolution. We report de novo-assembled AA-genome sequences for Oryza nivara, Oryza glaberrima, Oryza barthii, Oryza glumaepatula, and Oryza meridionalis. Our analyses reveal massive levels of genomic structural variation, including segmental duplication and rapid gene family turnover, with particularly high instability in defense-related genes. We show, on a genomic scale, how lineage-specific expansion or contraction of gene families has led to their morphological and reproductive diversification, thus enlightening the evolutionary process of speciation and adaptation. Despite strong purifying selective pressures on most Oryza genes, we documented a large number of positively selected genes, especially those genes involved in flower development, reproduction, and resistance-related processes. These diversifying genes are expected to have played key roles in adaptations to their ecological niches in Asia, South America, Africa and Australia. Extensive variation in noncoding RNA gene numbers, function enrichment, and rates of sequence divergence might also help account for the different genetic adaptations of these rice species. Collectively, these resources provide new opportunities for evolutionary genomics, numerous insights into recent speciation, a valuable database of functional variation for crop improvement, and tools for efficient conservation of wild rice germplasm. PMID:25368197
A study on biological activity of marine fungi from different habitats in coastal regions.

PubMed

Zhou, Songlin; Wang, Min; Feng, Qi; Lin, Yingying; Zhao, Huange

2016-01-01

In recent years, marine fungi have become an important source of active marine natural products. Former researches are limited in habitats selection of fungi with bioactive compounds. In this paper were to measure antibacterial and antitumor cell activity for secondary metabolites of marine fungi, which were isolated from different habitats in coastal regions. 195 strains of marine fungi were isolated and purified from three different habitats. They biologically active experiment results showed that fungi isolation from the mangrove habitats had stronger antibacterial activity than others, and the stains isolated from the estuarial habitats had the least antibacterial activity. However, the strains separated from beach habitats strongly inhibited tumor cell proliferation in vitro, and fungi of mangrove forest habitats had the weakest activity of inhibiting tumor. Meanwhile, 195 fungal strains belonged to 46 families, 84 genera, 142 species and also showed 137 different types of activity combinations by analyzing the inhibitory activity of the metabolites fungi for 4 strains of pathogenic bacteria and B-16 cells. The study investigated the biological activity of marine fungi isolated from different habitats in Haikou coastal regions. The results help us to understand bioactive metabolites of marine fungi from different habitats, and how to selected biological activity fungi from various marine habitats effectively.
Nucleotide diversity, natural variation, and evolution of Flexible culm-1 and Strong culm-2 lodging resistance genes in rice.

PubMed

Rashid, Muhammad Abdul Rehman; Zhao, Yan; Zhang, Hongliang; Li, Jinjie; Li, Zichao

2016-07-01

Lodging resistance is one of the vital traits in yield improvement and sustainability. Culm wall thickness, diameter, and strength are different traits that can govern the lodging resistance in rice. The genes SCM2 and FC1 have been isolated for culm thickness, strength, and flexibility, but their functional nucleotide variations were still unknown. We used a 13× deep sequence of 795 diverse genotypes to present the functional variation and SNP diversity in SCM2 and FC1. The major functional variant for the SCM2 gene was at position 27480181 and for the FC1 gene at position 31072992. Haplotype analysis of both genes provided their various allelic differences among haplotypes. SCM2 alleles further presented the evolution of Oryza sativa L. subsp. indica and subsp. japonica genomes from common parent in different geographical zones, while the haplotypes of FC1 suggested their evolution from different strains of the common parent Oryza rufipogon. SCM2 showed purifying selection and functional associations with rare alleles, while FC1 displayed balanced selection favored by multiple heterozygous alleles. Genotypes with an allelic combination of SCM2-3 and FC1-2 in japonica background exhibited striking resistance against lodging, which can be used in further breeding programs.
Comparative Genomics Reveals Accelerated Evolution in Conserved Pathways during the Diversification of Anole Lizards

PubMed Central

Tollis, Marc; Hutchins, Elizabeth D; Stapley, Jessica; Rupp, Shawn M; Eckalbar, Walter L; Maayan, Inbar; Lasku, Eris; Infante, Carlos R; Dennis, Stuart R; Robertson, Joel A; May, Catherine M; Bermingham, Eldredge; DeNardo, Dale F; Hsieh, Shi-Tong Tonia; Kulathinal, Rob J; McMillan, William Owen; Menke, Douglas B; Pratt, Stephen C; Rawls, Jeffery Alan; Sanjur, Oris; Wilson-Rawls, Jeanne; Wilson Sayres, Melissa A; Fisher, Rebecca E

2018-01-01

Abstract Squamates include all lizards and snakes, and display some of the most diverse and extreme morphological adaptations among vertebrates. However, compared with birds and mammals, relatively few resources exist for comparative genomic analyses of squamates, hampering efforts to understand the molecular bases of phenotypic diversification in such a speciose clade. In particular, the ∼400 species of anole lizard represent an extensive squamate radiation. Here, we sequence and assemble the draft genomes of three anole species—Anolis frenatus, Anolis auratus, and Anolis apletophallus—for comparison with the available reference genome of Anolis carolinensis. Comparative analyses reveal a rapid background rate of molecular evolution consistent with a model of punctuated equilibrium, and strong purifying selection on functional genomic elements in anoles. We find evidence for accelerated evolution in genes involved in behavior, sensory perception, and reproduction, as well as in genes regulating limb bud development and hindlimb specification. Morphometric analyses of anole fore and hindlimbs corroborated these findings. We detect signatures of positive selection across several genes related to the development and regulation of the forebrain, hormones, and the iguanian lizard dewlap, suggesting molecular changes underlying behavioral adaptations known to reinforce species boundaries were a key component in the diversification of anole lizards. PMID:29360978
Development and antigenic characterization of three recombinant proteins with potential for Glässer's disease prevention.

PubMed

Li, Miao; Li, Chunling; Song, Shuai; Kang, Huahua; Yang, Dongxia; Li, Guoqing

2016-04-27

Haemophilus parasuis is the causative agent of Glässer's disease, which causes high morbidity and mortality in piglets, leading to severe economic losses. The lack of a commercial vaccine against a broad spectrum of strains has limited the disease control. H. parasuis outer membrane proteins (OMPs) are potentially essential components for vaccine formulation. In this study, seven putative OMPs were selected from the annotated H. parasuis serovar 5 genome; they were predicted by bioinformatics and annotated as potential virulence-related factors. These proteins were cloned, expressed, and purified as His-tagged proteins. Antigenicity of the candidate proteins was assessed using Western blotting with convalescent sera against H. parasuis serovar 5. The immunogenicity of the seven OMPs was assessed in a guinea pig model. Except VacJ, all the other six recombinant proteins elicited a detectable antibody response. Antisera against four of the selected proteins effectively killed the bacteria in vitro. Three proteins (Omp26, VacJ, and HAPS_0742) were found to confer signiﬁcant protection against challenge with a lethal dose of H. parasuis in a guinea pig model. The results suggest that these three proteins have a strong potential to be vaccine candidates against Glässer's disease. Copyright © 2016 Elsevier Ltd. All rights reserved.

Genome-wide identification and characterization of Fox genes in the silkworm, Bombyx mori.

PubMed

Song, JiangBo; Li, ZhiQuan; Tong, XiaoLing; Chen, Cong; Chen, Min; Meng, Gang; Chen, Peng; Li, ChunLin; Xin, YaQun; Gai, TingTing; Dai, FangYin; Lu, Cheng

2015-09-01

The forkhead box (Fox) transcription factor family has a characteristic of forkhead domain, a winged DNA-binding domain. The Fox genes have been classified into 23 subfamilies, designated FoxA to FoxS, of which the FoxR and FoxS subfamilies are specific to vertebrates. In this review, using whole-genome scanning, we identified 17 distinct Fox genes distributed on 13 chromosomes of the silkworm, Bombyx mori. A phylogenetic tree showed that the silkworm Fox genes could be classified into 13 subfamilies. The FoxK subfamily is specifically absent from the silkworm, although it is present in other lepidopteran insects, including Danaus plexippus and Heliconius melpomene. Microarray data revealed that the Fox genes have distinct expression patterns in the tissues on day 3 of the 5th instar larva. A Gene Ontology analysis suggested that the Fox genes have roles in cellular components, molecular functions, and biological processes, except in pore complex biogenesis. An analysis of the selective pressure on the proteins indicated that most of the amino acid sites in the Fox proteins are undergoing strong purifying selection. Here, we summarize the general characteristics of the Fox genes in the silkworm, which should support further functional studies of the silkworm Fox proteins.
Conserved noncoding sequences conserve biological networks and influence genome evolution.

PubMed

Xie, Jianbo; Qian, Kecheng; Si, Jingna; Xiao, Liang; Ci, Dong; Zhang, Deqiang

2018-05-01

Comparative genomics approaches have identified numerous conserved cis-regulatory sequences near genes in plant genomes. Despite the identification of these conserved noncoding sequences (CNSs), our knowledge of their functional importance and selection remains limited. Here, we used a combination of DNA methylome analysis, microarray expression analyses, and functional annotation to study these sequences in the model tree Populus trichocarpa. Methylation in CG contexts and non-CG contexts was lower in CNSs, particularly CNSs in the 5'-upstream regions of genes, compared with other sites in the genome. We observed that CNSs are enriched in genes with transcription and binding functions, and this also associated with syntenic genes and those from whole-genome duplications, suggesting that cis-regulatory sequences play a key role in genome evolution. We detected a significant positive correlation between CNS number and protein interactions, suggesting that CNSs may have roles in the evolution and maintenance of biological networks. The divergence of CNSs indicates that duplication-degeneration-complementation drives the subfunctionalization of a proportion of duplicated genes from whole-genome duplication. Furthermore, population genomics confirmed that most CNSs are under strong purifying selection and only a small subset of CNSs shows evidence of adaptive evolution. These findings provide a foundation for future studies exploring these key genomic features in the maintenance of biological networks, local adaptation, and transcription.
Anti-hepatitis C virus activity and synergistic effect of Nymphaea alba extracts and bioactive constituents in liver infected cells.

PubMed

Rehman, Sidra; Ashfaq, Usman Ali; Ijaz, Bushra; Riazuddin, Sheikh

2018-05-28

Without an effective vaccine, hepatitis C virus (HCV) remains a global threat, inflicting 170-300 million carriers worldwide at risk of cirrhosis and hepatocellular carcinoma (HCC). Though various direct acting antivirals have been redeemed the hepatitis C treatment, a few restraints persist including possible side effects, viral resistance emergence, excessive cost which restricts its availability to a common person. There is no preventive HCV vaccine available today so the discovery of potent antiviral natural flora and their bioactive constituents may help to develop preventive cures against HCV infection. In current study, we aim to clarify anti-HCV activity of methanol and acetone extracts along with the purified fractions of Pakistani local plant, Nymphaea alba L (N. alba) using Huh-7 cell line as transfection model. Synergistic study of purified fractions with interferon was performed using MDBK cell line (expressing interferon receptors) as transfection model. Recent study by our research group has observed potent anti-HCV NS3 protease activity of methanol and acetone extracts of N. alba. Effect of N. alba extracts, its fractions precisely, the N1 and N8 fractions on HCV replication was demonstrated by analyzing viral gene expression using in vitro transfection model. Considering NS3 protease as a dynamic drug target, fourteen phytochemicals of N. alba were selected as ligands for interaction with NS3 protein using Molecular Operating Environment (MOE) software. Boceprevir, FDA approved NS3 protease inhibitor, was used as standard for comparative study in docking screening. Herein we report 84% and 94% reduction of 3a genotype of HCV NS3/4A gene expression at mRNA level at non-toxic concentration. Specifically, two fractions 'N1' & 'N8' isolated from acetone extract suppressed HCV NS3 gene expression in transfected target cells with an EC 50 value of 37 ± 0.03 μg/ml and 20 ± 0.02 μg/ml respectively. Similarly, viral genotype 1a replication is strongly suppressed in target cells by N. alba flower extracts and purified fractions. Moreover, combination of fractions with standard antiviral drug displayed synergistic effects for inhibition of HCV replication. Phytochemicals including Isoquercetin, Hyperoside, Quercetin, Reynoutrin, Apigenin and Isokaempferide displayed minimum binding energies as compared to standard protease inhibitor. N. alba and its purified phytochemicals with new scaffolds might significantly serve as valuable and alternative regimen against HCV either alone or in combination with other potential anti-HCV agents. Copyright © 2018 Elsevier Ltd. All rights reserved.
Molecular population genetics of the insulin/TOR signal transduction pathway: a network-level analysis in Drosophila melanogaster.

PubMed

Alvarez-Ponce, David; Guirao-Rico, Sara; Orengo, Dorcas J; Segarra, Carmen; Rozas, Julio; Aguadé, Montserrat

2012-01-01

The IT-insulin/target of rapamycin (TOR)-signal transduction pathway is a relatively well-characterized pathway that plays a central role in fundamental biological processes. Network-level analyses of DNA divergence in Drosophila and vertebrates have revealed a clear gradient in the levels of purifying selection along this pathway, with the downstream genes being the most constrained. Remarkably, this feature does not result from factors known to affect selective constraint such as gene expression, codon bias, protein length, and connectivity. The present work aims to establish whether the selective constraint gradient detected along the IT pathway at the between-species level can also be observed at a shorter time scale. With this purpose, we have surveyed DNA polymorphism in Drosophila melanogaster and divergence from D. simulans along the IT pathway. Our network-level analysis shows that DNA polymorphism exhibits the same polarity in the strength of purifying selection as previously detected at the divergence level. This equivalent feature detected both within species and between closely and distantly related species points to the action of a general mechanism, whose action is neither organism specific nor evolutionary time dependent. The detected polarity would be, therefore, intrinsic to the IT pathway architecture and function.
Efficient production of artificially designed gelatins with a Bacillus brevis system.

PubMed

Kajino, T; Takahashi, H; Hirai, M; Yamada, Y

2000-01-01

Artificially designed gelatins comprising tandemly repeated 30-amino-acid peptide units derived from human alphaI collagen were successfully produced with a Bacillus brevis system. The DNA encoding the peptide unit was synthesized by taking into consideration the codon usage of the host cells, but no clones having a tandemly repeated gene were obtained through the above-mentioned strategy. Minirepeat genes could be selected in vivo from a mixture of every possible sequence encoding an artificial gelatin by randomly ligating the mixed sequence unit and transforming it into Escherichia coli. Larger repeat genes constructed by connecting minirepeat genes obtained by in vivo selection were also stable in the expression host cells. Gelatins derived from the eight-unit and six-unit repeat genes were extracellularly produced at the level of 0.5 g/liter and easily purified by ammonium sulfate fractionation and anion-exchange chromatography. The purified artificial gelatins had the predicted N-terminal sequences and amino acid compositions and a solgel property similar to that of the native gelatin. These results suggest that the selection of a repeat unit sequence stable in an expression host is a shortcut for the efficient production of repetitive proteins and that it can conveniently be achieved by the in vivo selection method. This study revealed the possible industrial application of artificially designed repetitive proteins.
Cell selection and characterization of a novel human endothelial cell specific nanobody.

PubMed

Ahmadvand, Davoud; Rasaee, Mohammad J; Rahbarizadeh, Fatemeh; Kontermann, Roland E; Sheikholislami, Farzaneh

2009-05-01

Antibody-based targeting of angiogenesis and vascular targeting therapy of cancer are extremely attractive conceptually and open new important diagnostic and therapeutic opportunities. Compelling evidence suggests that CD105 represents an ideal target for anti-angiogenic therapy and its presence in solid tumor vasculature has prognostic value. Camelids produce functional antibodies devoid of light chains and constant heavy chain domain (CH1). Nanobodies, the antigen-binding fragments of such heavy chain antibodies, are therefore comprised in one single domain. The aim of this study was to explore the possibilities of using anti-endoglin nanobody as an angiogenesis inhibitor. The anti-CD105 nanobody (AR-86a) was isolated from immune library by selections on purified antigens and target cells. Immunocytochemistry and FACS analysis showed that the purified nanobody reacted specifically with human umbilical vein endothelial cells (HUVECs) but not with other cell lines such as MDA-MB-453, Mel III, T-47D, MCF-7, AGO and HT 29. Further, selected nanobody potently inhibited proliferation of human endothelial cells and formation of capillary-like structures. This selected high affinity anti-endoglin nanobody may offer high specificity towards tumors with reduced side effects, and may be less likely to elicit drug resistance compared to conventional therapy.
Parent-progeny sequencing indicates higher mutation rates in heterozygotes.

PubMed

Yang, Sihai; Wang, Long; Huang, Ju; Zhang, Xiaohui; Yuan, Yang; Chen, Jian-Qun; Hurst, Laurence D; Tian, Dacheng

2015-07-23

Mutation rates vary within genomes, but the causes of this remain unclear. As many prior inferences rely on methods that assume an absence of selection, potentially leading to artefactual results, we call mutation events directly using a parent-offspring sequencing strategy focusing on Arabidopsis and using rice and honey bee for replication. Here we show that mutation rates are higher in heterozygotes and in proximity to crossover events. A correlation between recombination rate and intraspecific diversity is in part owing to a higher mutation rate in domains of high recombination/diversity. Implicating diversity per se as a cause, we find an ∼3.5-fold higher mutation rate in heterozygotes than in homozygotes, with mutations occurring in closer proximity to heterozygous sites than expected by chance. In a genome that is a patchwork of heterozygous and homozygous domains, mutations occur disproportionately more often in the heterozygous domains. If segregating mutations predispose to a higher local mutation rate, clusters of genes dominantly under purifying selection (more commonly homozygous) and under balancing selection (more commonly heterozygous), might have low and high mutation rates, respectively. Our results are consistent with this, there being a ten times higher mutation rate in pathogen resistance genes, expected to be under positive or balancing selection. Consequently, we do not necessarily need to evoke extremely weak selection on the mutation rate to explain why mutational hot and cold spots might correspond to regions under positive/balancing and purifying selection, respectively.
Positive Selection in Rapidly Evolving Plastid–Nuclear Enzyme Complexes

PubMed Central

Rockenbach, Kate; Havird, Justin C.; Monroe, J. Grey; Triant, Deborah A.; Taylor, Douglas R.; Sloan, Daniel B.

2016-01-01

Rates of sequence evolution in plastid genomes are generally low, but numerous angiosperm lineages exhibit accelerated evolutionary rates in similar subsets of plastid genes. These genes include clpP1 and accD, which encode components of the caseinolytic protease (CLP) and acetyl-coA carboxylase (ACCase) complexes, respectively. Whether these extreme and repeated accelerations in rates of plastid genome evolution result from adaptive change in proteins (i.e., positive selection) or simply a loss of functional constraint (i.e., relaxed purifying selection) is a source of ongoing controversy. To address this, we have taken advantage of the multiple independent accelerations that have occurred within the genus Silene (Caryophyllaceae) by examining phylogenetic and population genetic variation in the nuclear genes that encode subunits of the CLP and ACCase complexes. We found that, in species with accelerated plastid genome evolution, the nuclear-encoded subunits in the CLP and ACCase complexes are also evolving rapidly, especially those involved in direct physical interactions with plastid-encoded proteins. A massive excess of nonsynonymous substitutions between species relative to levels of intraspecific polymorphism indicated a history of strong positive selection (particularly in CLP genes). Interestingly, however, some species are likely undergoing loss of the native (heteromeric) plastid ACCase and putative functional replacement by a duplicated cytosolic (homomeric) ACCase. Overall, the patterns of molecular evolution in these plastid–nuclear complexes are unusual for anciently conserved enzymes. They instead resemble cases of antagonistic coevolution between pathogens and host immune genes. We discuss a possible role of plastid–nuclear conflict as a novel cause of accelerated evolution. PMID:27707788
Evidence of pervasive biologically functional secondary structures within the genomes of eukaryotic single-stranded DNA viruses.

PubMed

Muhire, Brejnev Muhizi; Golden, Michael; Murrell, Ben; Lefeuvre, Pierre; Lett, Jean-Michel; Gray, Alistair; Poon, Art Y F; Ngandu, Nobubelo Kwanele; Semegni, Yves; Tanov, Emil Pavlov; Monjane, Adérito Luis; Harkins, Gordon William; Varsani, Arvind; Shepherd, Dionne Natalie; Martin, Darren Patrick

2014-02-01

Single-stranded DNA (ssDNA) viruses have genomes that are potentially capable of forming complex secondary structures through Watson-Crick base pairing between their constituent nucleotides. A few of the structural elements formed by such base pairings are, in fact, known to have important functions during the replication of many ssDNA viruses. Unknown, however, are (i) whether numerous additional ssDNA virus genomic structural elements predicted to exist by computational DNA folding methods actually exist and (ii) whether those structures that do exist have any biological relevance. We therefore computationally inferred lists of the most evolutionarily conserved structures within a diverse selection of animal- and plant-infecting ssDNA viruses drawn from the families Circoviridae, Anelloviridae, Parvoviridae, Nanoviridae, and Geminiviridae and analyzed these for evidence of natural selection favoring the maintenance of these structures. While we find evidence that is consistent with purifying selection being stronger at nucleotide sites that are predicted to be base paired than at sites predicted to be unpaired, we also find strong associations between sites that are predicted to pair with one another and site pairs that are apparently coevolving in a complementary fashion. Collectively, these results indicate that natural selection actively preserves much of the pervasive secondary structure that is evident within eukaryote-infecting ssDNA virus genomes and, therefore, that much of this structure is biologically functional. Lastly, we provide examples of various highly conserved but completely uncharacterized structural elements that likely have important functions within some of the ssDNA virus genomes analyzed here.
Evidence of Pervasive Biologically Functional Secondary Structures within the Genomes of Eukaryotic Single-Stranded DNA Viruses

PubMed Central

Muhire, Brejnev Muhizi; Golden, Michael; Murrell, Ben; Lefeuvre, Pierre; Lett, Jean-Michel; Gray, Alistair; Poon, Art Y. F.; Ngandu, Nobubelo Kwanele; Semegni, Yves; Tanov, Emil Pavlov; Monjane, Adérito Luis; Harkins, Gordon William; Varsani, Arvind; Shepherd, Dionne Natalie

2014-01-01

Single-stranded DNA (ssDNA) viruses have genomes that are potentially capable of forming complex secondary structures through Watson-Crick base pairing between their constituent nucleotides. A few of the structural elements formed by such base pairings are, in fact, known to have important functions during the replication of many ssDNA viruses. Unknown, however, are (i) whether numerous additional ssDNA virus genomic structural elements predicted to exist by computational DNA folding methods actually exist and (ii) whether those structures that do exist have any biological relevance. We therefore computationally inferred lists of the most evolutionarily conserved structures within a diverse selection of animal- and plant-infecting ssDNA viruses drawn from the families Circoviridae, Anelloviridae, Parvoviridae, Nanoviridae, and Geminiviridae and analyzed these for evidence of natural selection favoring the maintenance of these structures. While we find evidence that is consistent with purifying selection being stronger at nucleotide sites that are predicted to be base paired than at sites predicted to be unpaired, we also find strong associations between sites that are predicted to pair with one another and site pairs that are apparently coevolving in a complementary fashion. Collectively, these results indicate that natural selection actively preserves much of the pervasive secondary structure that is evident within eukaryote-infecting ssDNA virus genomes and, therefore, that much of this structure is biologically functional. Lastly, we provide examples of various highly conserved but completely uncharacterized structural elements that likely have important functions within some of the ssDNA virus genomes analyzed here. PMID:24284329
Selection and Trans-Species Polymorphism of Major Histocompatibility Complex Class II Genes in the Order Crocodylia

PubMed Central

Jaratlerdsiri, Weerachai; Isberg, Sally R.; Higgins, Damien P.; Miles, Lee G.; Gongora, Jaime

2014-01-01

Major Histocompatibility Complex (MHC) class II genes encode for molecules that aid in the presentation of antigens to helper T cells. MHC characterisation within and between major vertebrate taxa has shed light on the evolutionary mechanisms shaping the diversity within this genomic region, though little characterisation has been performed within the Order Crocodylia. Here we investigate the extent and effect of selective pressures and trans-species polymorphism on MHC class II α and β evolution among 20 extant species of Crocodylia. Selection detection analyses showed that diversifying selection influenced MHC class II β diversity, whilst diversity within MHC class II α is the result of strong purifying selection. Comparison of translated sequences between species revealed the presence of twelve trans-species polymorphisms, some of which appear to be specific to the genera Crocodylus and Caiman. Phylogenetic reconstruction clustered MHC class II α sequences into two major clades representing the families Crocodilidae and Alligatoridae. However, no further subdivision within these clades was evident and, based on the observation that most MHC class II α sequences shared the same trans-species polymorphisms, it is possible that they correspond to the same gene lineage across species. In contrast, phylogenetic analyses of MHC class II β sequences showed a mixture of subclades containing sequences from Crocodilidae and/or Alligatoridae, illustrating orthologous relationships among those genes. Interestingly, two of the subclades containing sequences from both Crocodilidae and Alligatoridae shared specific trans-species polymorphisms, suggesting that they may belong to ancient lineages pre-dating the divergence of these two families from the common ancestor 85–90 million years ago. The results presented herein provide an immunogenetic resource that may be used to further assess MHC diversity and functionality in Crocodylia. PMID:24503938
Genetic diversity and natural selection of Plasmodium knowlesi merozoite surface protein 1 paralog gene in Malaysia.

PubMed

Ahmed, Md Atique; Fauzi, Muh; Han, Eun-Taek

2018-03-14

Human infections due to the monkey malaria parasite Plasmodium knowlesi is on the rise in most Southeast Asian countries specifically Malaysia. The C-terminal 19 kDa domain of PvMSP1P is a potential vaccine candidate, however, no study has been conducted in the orthologous gene of P. knowlesi. This study investigates level of polymorphisms, haplotypes and natural selection of full-length pkmsp1p in clinical samples from Malaysia. A total of 36 full-length pkmsp1p sequences along with the reference H-strain and 40 C-terminal pkmsp1p sequences from clinical isolates of Malaysia were downloaded from published genomes. Genetic diversity, polymorphism, haplotype and natural selection were determined using DnaSP 5.10 and MEGA 5.0 software. Genealogical relationships were determined using haplotype network tree in NETWORK software v5.0. Population genetic differentiation index (F ST ) and population structure of parasite was determined using Arlequin v3.5 and STRUCTURE v2.3.4 software. Comparison of 36 full-length pkmsp1p sequences along with the H-strain identified 339 SNPs (175 non-synonymous and 164 synonymous substitutions). The nucleotide diversity across the full-length gene was low compared to its ortholog pvmsp1p. The nucleotide diversity was higher toward the N-terminal domains (pkmsp1p-83 and 30) compared to the C-terminal domains (pkmsp1p-38, 33 and 19). Phylogenetic analysis of full-length genes identified 2 distinct clusters of P. knowlesi from Malaysian Borneo. The 40 pkmsp1p-19 sequences showed low polymorphisms with 16 polymorphisms leading to 18 haplotypes. In total there were 10 synonymous and 6 non-synonymous substitutions and 12 cysteine residues were intact within the two EGF domains. Evidence of strong purifying selection was observed within the full-length sequences as well in all the domains. Shared haplotypes of 40 pkmsp1p-19 were identified within Malaysian Borneo haplotypes. This study is the first to report on the genetic diversity and natural selection of pkmsp1p. A low level of genetic diversity and strong evidence of negative selection was detected and observed in all the domains of pkmsp1p of P. knowlesi indicating functional constrains. Shared haplotypes were identified within pkmsp1p-19 highlighting further evaluation using larger number of clinical samples from Malaysia.
Human scFv antibodies (Afribumabs) against Africanized bee venom: Advances in melittin recognition.

PubMed

Pessenda, Gabriela; Silva, Luciano C; Campos, Lucas B; Pacello, Elenice M; Pucca, Manuela B; Martinez, Edson Z; Barbosa, José E

2016-03-15

Africanized Apis mellifera bees, also known as killer bees, have an exceptional defensive instinct, characterized by mass attacks that may cause envenomation or death. From the years 2000-2013, 77,066 bee accidents occurred in Brazil. Bee venom comprises several substances, including melittin and phospholipase A2 (PLA2). Due to the lack of antivenom for bee envenomation, this study aimed to produce human monoclonal antibody fragments (single chain fragment variable; scFv), by using phage display technology. These fragments targeted melittin and PLA2, the two major components of bee venom, to minimize their toxic effects in cases of mass envenomation. Two phage antibody selections were performed using purified melittin. As the commercial melittin is contaminated with PLA2, phages specific to PLA2 were also obtained during one of the selections. Specific clones for melittin and PLA2 were selected for the production of soluble scFvs, named here Afribumabs: prefix: afrib- (from Africanized bee); stem/suffix: -umab (fully human antibody). Afribumabs 1 and 2 were tested in in vitro and in vivo assays to assess their ability to inhibit the toxic actions of purified melittin, PLA2, and crude bee venom. Afribumabs reduced hemolysis caused by purified melittin and PLA2 and by crude venom in vitro and reduced edema formation in the paws of mice and prolonged the survival of venom-injected animals in vivo. These results demonstrate that Afribumabs may contribute to the production of the first non-heterologous antivenom treatment against bee envenomation. Such a treatment may overcome some of the difficulties associated with conventional immunotherapy techniques. Copyright © 2016 Elsevier Ltd. All rights reserved.
Expression and purification of recombinant human serum albumin from selectively terminable transgenic rice.

PubMed

Zhang, Qing; Yu, Hui; Zhang, Feng-zhen; Shen, Zhi-cheng

2013-10-01

Human serum albumin (HSA) is widely utilized for medical purposes and biochemical research. Transgenic rice has proved to be an attractive bioreactor for mass production of recombinant HSA (rHSA). However, transgene spread is a major environmental and food safety concern for transgenic rice expressing proteins of medical value. This study aimed to develop a selectively terminable transgenic rice line expressing HSA in rice seeds, and a simple process for recovery and purification of rHSA for economical manufacture. An HSA expression cassette was inserted into a T-DNA vector encoding an RNA interference (RNAi) cassette suppressing the CYP81A6 gene. This gene detoxifies the herbicide bentazon and is linked to the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) cassette which confers glyphosate tolerance. ANX Sepharose Fast Flow (ANX FF) anion exchange chromatography coupled with Butyl Sepharose High Performance (Butyl HP) hydrophobic interaction chromatography was used to purify rHSA. A transgenic rice line, HSA-84, was obtained with stable expression of rHSA of up to 0.72% of the total dry weight of the dehusked rice seeds. This line also demonstrated high sensitivity to bentazon, and thus could be killed selectively by a spray of bentazon. A two-step chromatography purification scheme was established to purify the rHSA from rice seeds to a purity of 99% with a recovery of 62.4%. Results from mass spectrometry and N-terminus sequencing suggested that the purified rHSA was identical to natural plasma-derived HSA. This study provides an alternative strategy for large-scale production of HSA with a built-in transgene safety control mechanism.
Solubilization conditions for bovine heart mitochondrial membranes allow selective purification of large quantities of respiratory complexes I, III, and V.

PubMed

Shimada, Satoru; Maeda, Shintaro; Hikita, Masahide; Mieda-Higa, Kaoru; Uene, Shigefumi; Nariai, Yukiko; Shinzawa-Itoh, Kyoko

2018-04-24

Ascertaining the structure and functions of mitochondrial respiratory chain complexes is essential to understanding the biological mechanisms of energy conversion; therefore, numerous studies have examined these complexes. A fundamental part of that research involves devising a method for purifying samples with good reproducibility; the samples obtained need to be stable and their constituents need to retain the same structure and functions they possess when in mitochondrial membranes. Submitochondrial bovine heart particles were isolated using differential centrifugation to adjust to a membrane concentration of 46.0% (w/v) or 31.5% (w/v) based on weight. After 0.7% (w/v) deoxycholic acid, 0.4% (w/v) decyl maltoside, and 7.2% (w/v) potassium chloride were added to the mitochondrial membranes, those membranes were solubilized. At a membrane concentration of 46%, complex V was selectively solubilized, whereas at a concentration of 31.5% (w/v), complexes I and III were solubilized. Two steps-sucrose density gradient centrifugation and anion-exchange chromatography on a POROS HQ 20 μm column-enabled selective purification of samples that retained their structure and functions. These two steps enabled complexes I, III, and V to be purified in two days with a high yield. Complexes I, III, and V were stabilized with n-decyl-β-D-maltoside. A total of 200 mg-300 mg of those complexes from one bovine heart (1.1 kg muscle) was purified with good reproducibility, and the complexes retained the same functions they possessed while in mitochondrial membranes. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.
Expression and purification of native and functional influenza A virus matrix 2 proton selective ion channel.

PubMed

Desuzinges Mandon, Elodie; Traversier, Aurélien; Champagne, Anne; Benier, Lorraine; Audebert, Stéphane; Balme, Sébastien; Dejean, Emmanuel; Rosa Calatrava, Manuel; Jawhari, Anass

2017-03-01

Influenza A virus displays one of the highest infection rates of all human viruses and therefore represents a severe human health threat associated with an important economical challenge. Influenza matrix protein 2 (M2) is a membrane protein of the viral envelope that forms a proton selective ion channel. Here we report the expression and native isolation of full length active M2 without mutations or fusions. The ability of the influenza virus to efficiently infect MDCK cells was used to express native M2 protein. Using a Calixarene detergents/surfactants based approach; we were able to solubilize most of M2 from the plasma membrane and purify it. The tetrameric form of native M2 was maintained during the protein preparation. Mass spectrometry shows that M2 was phosphorylated in its cytoplasmic tail (serine 64) and newly identifies an acetylation of the highly conserved Lysine 60. ELISA shows that solubilized and purified M2 was specifically recognized by M2 antibody MAB65 and was able to displace the antibody from M2 MDCK membranes. Using a bilayer voltage clamp measurement assay, we demonstrate a pH dependent proton selective ion channel activity. The addition of the M2 ion channel blocker amantadine allows a total inhibition of the channel activity, illustrating therefore the specificity of purified M2 activity. Taken together, this work shows the production and isolation of a tetrameric and functional native M2 ion channel that will pave the way to structural and functional characterization of native M2, conformational antibody development, small molecules compounds screening towards vaccine treatment. Copyright © 2016 Elsevier Inc. All rights reserved.
Characterization of polysaccharides with marked inhibitory effect on lipid accumulation in Pleurotus eryngii.

PubMed

Chen, Jingjing; Yong, Yangyang; Xing, Meichun; Gu, Yifan; Zhang, Zhao; Zhang, Shizhu; Lu, Ling

2013-09-12

Mushrooms have a great potential for the production of useful bioactive metabolites. To explore the bioactive compounds from edible mushrooms for interfering with the development of macrophage-derived foam cells, which is recognized as the hallmark of early atherosclerosis, eight types of mushrooms polysaccharides had been selected to be tested. Consequently, different mushrooms polysaccharides displayed diverse component profiles. Of polysaccharides that we tested, the Pleurotus eryngii polysaccharide had the strongest inhibitory effect on lipid accumulation. Furthermore, through fractionation of DEAE-52 and Sephadex G-100, the polysaccharide from P. eryngii had been successfully purified and identified. By the analysis of IR, GC, and HPLC, the purified polysaccharide was estimated to be 30-38 kDa for the average molecular weight with the monosaccharide composition mainly composed of D-types of mannose, glucose and galactose. Findings presented in this report firstly provide direct evidence, which links the purified polysaccharide moiety with the biological function in foam-cell model. Copyright © 2013 Elsevier Ltd. All rights reserved.
Human proinsulin C-peptide from a precursor overexpressed in Pichia pastoris.

PubMed

Huang, Yang-Bin; Li, Jiang; Gao, Xin; Sun, Jiu-Ru; Lu, Yi; Feng, Tao; Fei, Jian; Cui, Da-Fu; Xia, Qi-Chang; Ren, Jun; Zhang, You-Shang

2006-08-01

In this article we report the production of human proinsulin C-peptide with 31 amino acid residues from a precursor overexpressed in Pichia pastoris. A C-peptide precursor expression plasmid containing nine C-peptide genes in tandem was constructed and used to transform P. pastoris. Transformants with a high copy number of the C-peptide precursor gene integrated into the chromosome of P. pastoris were selected. In high-density fermentation in a 300 liter fermentor using a simple culture medium composed mainly of salt and methanol, the C-peptide precursor was overexpressed to a level of 2.28 g per liter. A simple procedure was established to purify the expression product from the culture medium. The purified C-peptide precursor was converted into C-peptide by trypsin and carboxypeptidase B joint digestion. The yield of C-peptide with a purity of 96% was 730 mg per liter of culture. The purified C-peptide was characterized by mass spectrometry, N- and C-terminal amino acid sequencing, and sodium dodecylsulfate-polyacrylamide gel electrophoresis.
Protection from Staphylococcus aureus mastitis associated with poly-N-acetyl β-1,6 glucosamine specific antibody production using biofilm-embedded bacteria

PubMed Central

Pérez, M. M.; Prenafeta, A.; Valle, J.; Penadés, J.; Rota, C.; Solano, C.; Marco, J.; Grilló, M.J.; Lasa, I.; Irache, J.M.; Maira-Litran, T.; Jiménez-Barbero, J.; Costa, L.; Pier, G.B.; de Andrés, D.; Amorena, B.

2010-01-01

Staphylococcus aureus vaccines based on bacterins surrounded by slime, surface polysaccharides coupled to protein carriers and polysaccharides embedded in liposomes administered together with non-biofilm bacterins confer protection against mastitis. However, it remains unknown whether protective antibodies are directed to slime-associated known exopolysaccharides and could be produced in the absence of bacterin immunizations. Here, a sheep mastitis vaccination study was carried out using bacterins, crude bacterial extracts or a purified exopolysaccharide from biofilm bacteria delivered in different vehicles. This polysaccharide reacted specifically with antibodies to poly-N-acetyl-β-1,6-glucosamine (PNAG) and not with antibodies to other capsular antigens or bacterial components. Following intra-mammary challenge with biofilm-producing bacteria, antibody production against the polysaccharide, milk bacterial counts and mastitis lesions were determined. Bacterins from strong biofilm-producing bacteria triggered the highest production of antibodies to PNAG and conferred the highest protection against infection and mastitis, compared with weak biofilm-producing bacteria and non-cellular inocula. Thus, bacterins from strong biofilm bacteria, rather than purified polysaccharide, are proposed as a cost-efficient vaccination against S. aureus ruminant mastitis. PMID:19428854
Partial purification and characterization of polyphenol oxidase from banana (Musa sapientum L.) peel.

PubMed

Yang, C P; Fujita, S; Kohno, K; Kusubayashi, A; Ashrafuzzaman, M; Hayashi, N

2001-03-01

Polyphenol oxidase (EC 1.10.3.1, o-diphenol: oxygen oxidoreductase, PPO) of banana (Musa sapientum L.) peel was partially purified about 460-fold with a recovery of 2.2% using dopamine as substrate. The enzyme showed a single peak on Toyopearl HW55-S chromatography. However, two bands were detected by staining with Coomassie brilliant blue on PAGE: one was very clear, and the other was faint. Molecular weight for purified PPO was estimated to be about 41 000 by gel filtration. The enzyme quickly oxidized dopamine, and its Km value (Michaelis constant) for dopamine was 3.9 mM. Optimum pH was 6.5 and the PPO activity was quite stable in the range of pH 5-11 for 48 h. The enzyme had an optimum temperature at 30 degrees C and was stable up to 60 degrees C after heat treatment for 30 min. The enzyme activity was strongly inhibited by sodium diethyldithiocarbamate, potassium cyanide, L-ascorbic acid, and cysteine at 1 mM. Under a low buffer capacity, the enzyme was also strongly inhibited by citric acid and acetic acid at 10 mM.

Characterization of a Thermostable d-Stereospecific Alanine Amidase from Brevibacillus borstelensis BCS-1

PubMed Central

Baek, Dae Heoun; Kwon, Seok-Joon; Hong, Seung-Pyo; Kwak, Mi-Sun; Lee, Mi-Hwa; Song, Jae Jun; Lee, Seung-Goo; Yoon, Ki-Hong; Sung, Moon-Hee

2003-01-01

A gene encoding a new thermostable d-stereospecific alanine amidase from the thermophile Brevibacillus borstelensis BCS-1 was cloned and sequenced. The molecular mass of the purified enzyme was estimated to be 199 kDa after gel filtration chromatography and about 30 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating that the enzyme could be composed of a hexamer with identical subunits. The purified enzyme exhibited strong amidase activity towards d-amino acid-containing aromatic, aliphatic, and branched amino acid amides yet exhibited no enzyme activity towards l-amino acid amides, d-amino acid-containing peptides, and NH2-terminally protected amino acid amides. The optimum temperature and pH for the enzyme activity were 85°C and 9.0, respectively. The enzyme remained stable within a broad pH range from 7.0 to 10.0. The enzyme was inhibited by dithiothreitol, 2-mercaptoethanol, and EDTA yet was strongly activated by Co2+ and Mn2+. The kcat/Km for d-alaninamide was measured as 544.4 ± 5.5 mM−1 min−1 at 50°C with 1 mM Co2+. PMID:12571020
Purification and characterization of a novel thermostable luciferase from Benthosema pterotum.

PubMed

Homaei, Ahmad Abolpour; Mymandi, Asma Bahari; Sariri, Reyhaneh; Kamrani, Ehsan; Stevanato, Roberto; Etezad, Seyed-Masoud; Khajeh, Khosro

2013-08-05

A novel luciferase from Benthosema pterotum, collected from Port of Jask, close to Persian Gulf, was purified for the first time, using Q-Sepharose anion exchange chromatography. The molecular mass of the novel enzyme, measured by SDS-PAGE technique, was about 27 kDa and its Km value is 0.4 μM; both values are similar to those of other coelenterazine luciferases. B. pterotum (BP) luciferase showed maximum intensity of emitted light at 40°C, in 20mM Tris buffer, pH 9 and 20 mM magnesium concentration. Experimental measurements indicated that BP luciferase is a relatively thermostable enzyme; furthermore it shows a high residual activity at extreme pH values. Its biological activity is strongly inhibited by 1 mM Cu(2+), Zn(2+) and Ni(2+), while calcium and mainly magnesium ions strongly increase BP luciferase activity. The B. pterotum luciferase generated blue light with a maximum emission wavelength at 475 nm and showed some similarity with other luciferases, while other parameters appeared quite different, in this way, confirming that a novel protein has been purified. Copyright © 2013 Elsevier B.V. All rights reserved.
Molecular phylogenetic and evolutionary analyses of Muar strain of Japanese encephalitis virus reveal it is the missing fifth genotype.

PubMed

Mohammed, Manal A F; Galbraith, Sareen E; Radford, Alan D; Dove, Winifred; Takasaki, Tomohiko; Kurane, Ichiro; Solomon, Tom

2011-07-01

Japanese encephalitis virus (JEV) is the most important cause of epidemic encephalitis worldwide but its origin is unknown. Epidemics of encephalitis suggestive of Japanese encephalitis (JE) were described in Japan from the 1870s onwards. Four genotypes of JEV have been characterised and representatives of each genotype have been fully sequenced. Based on limited information, a single isolate from Malaysia is thought to represent a putative fifth genotype. We have determined the complete nucleotide and amino acid sequence of Muar strain and compared it with other fully sequenced JEV genomes. Muar was the least similar, with nucleotide divergence ranging from 20.2 to 21.2% and amino acid divergence ranging from 8.5 to 9.9%. Phylogenetic analysis of Muar strain revealed that it does represent a distinct fifth genotype of JEV. We elucidated Muar signature amino acids in the envelope (E) protein, including E327 Glu on the exposed lateral surface of the putative receptor binding domain which distinguishes Muar strain from the other four genotypes. Evolutionary analysis of full-length JEV genomes revealed that the mean evolutionary rate is 4.35 × 10(-4) (3.4906 × 10(-4) to 5.303 × 10(-4)) nucleotides substitutions per site per year and suggests JEV originated from its ancestral virus in the mid 1500s in the Indonesia-Malaysia region and evolved there into different genotypes, which then spread across Asia. No strong evidence for positive selection was found between JEV strains of the five genotypes and the E gene has generally been subjected to strong purifying selection. Copyright © 2011 Elsevier B.V. All rights reserved.
Persistence of Lineage IV Peste-des-petits ruminants virus within Israel since 1993: An evolutionary perspective

PubMed Central

Clarke, Brian; Mahapatra, Mana; Friedgut, Orly; Bumbarov, Velizar

2017-01-01

Peste-des-petits ruminants (PPR) is one of the most important infectious diseases of domesticated small ruminants. From the initial identification in 1942 in West Africa, PPR virus (PPRV) has spread throughout much of the developing world. PPRV is now considered endemic throughout Africa, with the notable exception of South Africa, the Middle-East and Israel, as well as South-, East-, and Central Asia. Despite this widespread dispersal, the evolution and transmission of PPRV in endemic populations is not well understood. This understanding will be critical in the planning of rational measures to eradicate PPRV by the planned time as defined by the FAO and OIE. To further advance the understanding of the evolution of PPRV the full genome sequence of 18 viruses isolated from Israel from consecutive years between 1997–2014 were generated. This data set is unique and crucial for the understanding of the evolution of PPRV, as it represents the first set of full-length sequence data available from consecutive years from a single geographic location. Analysis of these full genome sequences shows 96.2–99.9% nucleotide conservation across the Israel isolates and further demonstrates the strong purifying selection pressures on PPRV within Israel and globally. Four amino acid substitutions indicative of putative positive selection were additionally identified within the Israel isolates. The mean substitution rate per site per year was estimated to be 9.22 x 10−4 (95% HPD 6.206 x 10−4–1.26 x 10−3). Using Bayesian and phylogenetic analyses we further demonstrate that the PPRV isolates from Israel belongs to linage IV and form a single strong regional cluster within all other lineage IV viruses circulating worldwide implying a single incursion into Israel. PMID:28545149
Persistence of Lineage IV Peste-des-petits ruminants virus within Israel since 1993: An evolutionary perspective.

PubMed

Clarke, Brian; Mahapatra, Mana; Friedgut, Orly; Bumbarov, Velizar; Parida, Satya

2017-01-01

Peste-des-petits ruminants (PPR) is one of the most important infectious diseases of domesticated small ruminants. From the initial identification in 1942 in West Africa, PPR virus (PPRV) has spread throughout much of the developing world. PPRV is now considered endemic throughout Africa, with the notable exception of South Africa, the Middle-East and Israel, as well as South-, East-, and Central Asia. Despite this widespread dispersal, the evolution and transmission of PPRV in endemic populations is not well understood. This understanding will be critical in the planning of rational measures to eradicate PPRV by the planned time as defined by the FAO and OIE. To further advance the understanding of the evolution of PPRV the full genome sequence of 18 viruses isolated from Israel from consecutive years between 1997-2014 were generated. This data set is unique and crucial for the understanding of the evolution of PPRV, as it represents the first set of full-length sequence data available from consecutive years from a single geographic location. Analysis of these full genome sequences shows 96.2-99.9% nucleotide conservation across the Israel isolates and further demonstrates the strong purifying selection pressures on PPRV within Israel and globally. Four amino acid substitutions indicative of putative positive selection were additionally identified within the Israel isolates. The mean substitution rate per site per year was estimated to be 9.22 x 10-4 (95% HPD 6.206 x 10-4-1.26 x 10-3). Using Bayesian and phylogenetic analyses we further demonstrate that the PPRV isolates from Israel belongs to linage IV and form a single strong regional cluster within all other lineage IV viruses circulating worldwide implying a single incursion into Israel.
Adaptive Evolution of Eel Fluorescent Proteins from Fatty Acid Binding Proteins Produces Bright Fluorescence in the Marine Environment.

PubMed

Gruber, David F; Gaffney, Jean P; Mehr, Shaadi; DeSalle, Rob; Sparks, John S; Platisa, Jelena; Pieribone, Vincent A

2015-01-01

We report the identification and characterization of two new members of a family of bilirubin-inducible fluorescent proteins (FPs) from marine chlopsid eels and demonstrate a key region of the sequence that serves as an evolutionary switch from non-fluorescent to fluorescent fatty acid-binding proteins (FABPs). Using transcriptomic analysis of two species of brightly fluorescent Kaupichthys eels (Kaupichthys hyoproroides and Kaupichthys n. sp.), two new FPs were identified, cloned and characterized (Chlopsid FP I and Chlopsid FP II). We then performed phylogenetic analysis on 210 FABPs, spanning 16 vertebrate orders, and including 163 vertebrate taxa. We show that the fluorescent FPs diverged as a protein family and are the sister group to brain FABPs. Our results indicate that the evolution of this family involved at least three gene duplication events. We show that fluorescent FABPs possess a unique, conserved tripeptide Gly-Pro-Pro sequence motif, which is not found in non-fluorescent fatty acid binding proteins. This motif arose from a duplication event of the FABP brain isoforms and was under strong purifying selection, leading to the classification of this new FP family. Residues adjacent to the motif are under strong positive selection, suggesting a further refinement of the eel protein's fluorescent properties. We present a phylogenetic reconstruction of this emerging FP family and describe additional fluorescent FABP members from groups of distantly related eels. The elucidation of this class of fish FPs with diverse properties provides new templates for the development of protein-based fluorescent tools. The evolutionary adaptation from fatty acid-binding proteins to fluorescent fatty acid-binding proteins raises intrigue as to the functional role of bright green fluorescence in this cryptic genus of reclusive eels that inhabit a blue, nearly monochromatic, marine environment.
Comparative Mitogenomic Analysis of Species Representing Six Subfamilies in the Family Tenebrionidae

PubMed Central

Zhang, Hong-Li; Liu, Bing-Bing; Wang, Xiao-Yang; Han, Zhi-Ping; Zhang, Dong-Xu; Su, Cai-Na

2016-01-01

To better understand the architecture and evolution of the mitochondrial genome (mitogenome), mitogenomes of ten specimens representing six subfamilies in Tenebrionidae were selected, and comparative analysis of these mitogenomes was carried out in this study. Ten mitogenomes in this family share a similar gene composition, gene order, nucleotide composition, and codon usage. In addition, our results show that nucleotide bias was strongly influenced by the preference of codon usage for A/T rich codons which significantly correlated with the G + C content of protein coding genes (PCGs). Evolutionary rate analyses reveal that all PCGs have been subjected to a purifying selection, whereas 13 PCGs displayed different evolution rates, among which ATPase subunit 8 (ATP8) showed the highest evolutionary rate. We inferred the secondary structure for all RNA genes of Tenebrio molitor (Te2) and used this as the basis for comparison with the same genes from other Tenebrionidae mitogenomes. Some conserved helices (stems) and loops of RNA structures were found in different domains of ribosomal RNAs (rRNAs) and the cloverleaf structure of transfer RNAs (tRNAs). With regard to the AT-rich region, we analyzed tandem repeat sequences located in this region and identified some essential elements including T stretches, the consensus motif at the flanking regions of T stretch, and the secondary structure formed by the motif at the 3′ end of T stretch in major strand, which are highly conserved in these species. Furthermore, phylogenetic analyses using mitogenomic data strongly support the relationships among six subfamilies: ((Tenebrionidae incertae sedis + (Diaperinae + Tenebrioninae)) + (Pimeliinae + Lagriinae)), which is consistent with phylogenetic results based on morphological traits. PMID:27258256
Evidence for a purifying selection acting on the β-lactamase locus in epidemic clones of methicillin-resistant Staphylococcus aureus.

PubMed

Milheiriço, Catarina; Portelinha, Ana; Krippahl, Ludwig; de Lencastre, Hermínia; Oliveira, Duarte C

2011-04-15

The β-lactamase (bla) locus, which confers resistance to penicillins only, may control the transcription of mecA, the central element of methicillin resistance, which is embedded in a polymorphic heterelogous chromosomal cassette (the SCCmec element). In order to assess the eventual correlation between bla allotypes and genetic lineages, SCCmec types and/or β-lactam resistance phenotypes, the allelic variation on the bla locus was evaluated in a representative collection of 54 international epidemic methicillin-resistant Staphylococcus aureus (MRSA) clinical strains and, for comparative purposes, also in 24 diverse methicillin-susceptible S. aureus (MSSA) strains. Internal fragments of blaZ (the β-lactamase structural gene) were sequenced for all strains. A subset of strains, representative of blaZ allotypes, was further characterized by sequencing of internal fragments of the blaZ transcriptional regulators, blaI and blaR1. Thirteen allotypes for blaZ, nine for blaI and 12 for blaR1 were found. In a total of 121 unique single-nucleotide polymorphisms (SNP) detected, no frameshift mutations were identified and only one nonsense mutation within blaZ was found in a MRSA strain. On average, blaZ alleles were more polymorphic among MSSA than in MRSA (14.7 vs 11.4 SNP/allele). Overall, blaR1 was the most polymorphic gene with an average of 24.8 SNP/allele. No correlation could be established between bla allotypes and genetic lineages, SCCmec types and/or β-lactam resistance phenotypes. In order to estimate the selection pressure acting on the bla locus, the average dN/dS values were computed. In the three genes and in both collections dN/dS ratios were significantly below 1. The data strongly suggests the existence of a purifying selection to maintain the bla locus fully functional even on MRSA strains. Although, this is in agreement with the notion that in most clinical MRSA strains mecA gene is under the control of the bla regulatory genes, these findings also suggest that the apparently redundant function of blaZ gene for the MRSA resistant phenotype is still important for these strains. In addition, the data shows that the sensor-inducer blaR1 is the primary target for the accumulation of mutations in the bla locus, presumably to modulate the response to the presence of β-lactam antibiotic.
Efficient syntheses of polyamine and polyamine amide voltage-sensitive calcium channel blockers: FTX-3.3 and sFTX-3.3.

PubMed

Moya, E; Blagbrough, I S

1996-02-01

Efficient syntheses of FTX-3.3 and sFTX-3.3, voltage-sensitive calcium channel blockers are described. These modified polyamines were prepared from selectively protected polyamines and purified on a practical scale.
Size-selective separation of polydisperse gold nanoparticles in supercritical ethane.

PubMed

Williams, Dylan P; Satherley, John

2009-04-09

The aim of this study was to use supercritical ethane to selectively disperse alkanethiol-stabilized gold nanoparticles of one size from a polydisperse sample in order to recover a monodisperse fraction of the nanoparticles. A disperse sample of metal nanoparticles with diameters in the range of 1-5 nm was prepared using established techniques then further purified by Soxhlet extraction. The purified sample was subjected to supercritical ethane at a temperature of 318 K in the pressure range 50-276 bar. Particles were characterized by UV-vis absorption spectroscopy, TEM, and MALDI-TOF mass spectroscopy. The results show that with increasing pressure the dispersibility of the nanoparticles increases, this effect is most pronounced for smaller nanoparticles. At the highest pressure investigated a sample of the particles was effectively stripped of all the smaller particles leaving a monodisperse sample. The relationship between dispersibility and supercritical fluid density for two different size samples of alkanethiol-stabilized gold nanoparticles was considered using the Chrastil chemical equilibrium model.
Determination of zearalenone and its metabolites in endometrial cancer by coupled separation techniques.

PubMed

Gadzała-Kopciuch, Renata; Cendrowski, Krzysztof; Cesarz, Anna; Kiełbasa, Paweł; Buszewski, Bogusław

2011-10-01

This study presents a selective method of isolation of zearalenone (ZON) and its metabolite, α-zearalenol (α-ZOL), in neoplastically changed human tissue by accelerated solvent and ultrasonic extractions using a mixture of acetonitrile/water (84/16% v/v) as the extraction solvent. Extraction effectiveness was determined through the selection of parameters (composition of the solvent mixture, temperature, pressure, number of cycles) with tissue contamination at the level of nanograms per gram. The produced acetonitrile/water extracts were purified, and analytes were enriched in columns packed with homemade molecularly imprinted polymers. Purified extracts were determined by liquid chromatography (LC) coupled with different detection systems (diode array detection--DAD and mass spectrometry--MS) involving the Ascentis RP-Amide as a stationary phase and gradient elution. The combination of UE-MISPE-LC (ultrasonic extraction--molecularly imprinted solid-phase extraction--liquid chromatography) produced high (R≈95-98%) and repeatable (RSD<3%) recovery values for ZON and α-ZOL. © The Author(s) 2011. This article is published with open access at Springerlink.com
Commercial Milk Enzyme-Linked Immunosorbent Assay (ELISA) Kit Reactivities to Purified Milk Proteins and Milk-Derived Ingredients.

PubMed

Ivens, Katherine O; Baumert, Joseph L; Taylor, Steve L

2016-07-01

Numerous commercial enzyme-linked immunosorbent assay (ELISA) kits exist to quantitatively detect bovine milk residues in foods. Milk contains many proteins that can serve as ELISA targets including caseins (α-, β-, or κ-casein) and whey proteins (α-lactalbumin or β-lactoglobulin). Nine commercially-available milk ELISA kits were selected to compare the specificity and sensitivity with 5 purified milk proteins and 3 milk-derived ingredients. All of the milk kits were capable of quantifying nonfat dry milk (NFDM), but did not necessarily detect all individual protein fractions. While milk-derived ingredients were detected by the kits, their quantitation may be inaccurate due to the use of different calibrators, reference materials, and antibodies in kit development. The establishment of a standard reference material for the calibration of milk ELISA kits is increasingly important. The appropriate selection and understanding of milk ELISA kits for food analysis is critical to accurate quantification of milk residues and informed risk management decisions. © 2016 Institute of Food Technologists®
Reduced MHC and neutral variation in the Galápagos hawk, an island endemic

PubMed Central

2011-01-01

Background Genes at the major histocompatibility complex (MHC) are known for high levels of polymorphism maintained by balancing selection. In small or bottlenecked populations, however, genetic drift may be strong enough to overwhelm the effect of balancing selection, resulting in reduced MHC variability. In this study we investigated MHC evolution in two recently diverged bird species: the endemic Galápagos hawk (Buteo galapagoensis), which occurs in small, isolated island populations, and its widespread mainland relative, the Swainson's hawk (B. swainsoni). Results We amplified at least two MHC class II B gene copies in each species. We recovered only three different sequences from 32 Galápagos hawks, while we amplified 20 unique sequences in 20 Swainson's hawks. Most of the sequences clustered into two groups in a phylogenetic network, with one group likely representing pseudogenes or nonclassical loci. Neutral genetic diversity at 17 microsatellite loci was also reduced in the Galápagos hawk compared to the Swainson's hawk. Conclusions The corresponding loss in neutral diversity suggests that the reduced variability present at Galápagos hawk MHC class II B genes compared to the Swainson's hawk is primarily due to a founder event followed by ongoing genetic drift in small populations. However, purifying selection could also explain the low number of MHC alleles present. This lack of variation at genes involved in the adaptive immune response could be cause for concern should novel diseases reach the archipelago. PMID:21612651
Molecular epidemiology of Powassan virus in North America.

PubMed

Pesko, Kendra N; Torres-Perez, Fernando; Hjelle, Brian L; Ebel, Gregory D

2010-11-01

Powassan virus (POW) is a tick-borne flavivirus distributed in Canada, the northern USA and the Primorsky region of Russia. POW is the only tick-borne flavivirus endemic to the western hemisphere, where it is transmitted mainly between Ixodes cookei and groundhogs (Marmota monax). Deer tick virus (DTV), a genotype of POW that has been frequently isolated from deer ticks (Ixodes scapularis), appears to be maintained in an enzootic cycle between these ticks and white-footed mice (Peromyscus leucopus). DTV has been isolated from ticks in several regions of North America, including the upper Midwest and the eastern seaboard. The incidence of human disease due to POW is apparently increasing. Previous analysis of tick-borne flaviviruses endemic to North America have been limited to relatively short genome fragments. We therefore assessed the evolutionary dynamics of POW using newly generated complete and partial genome sequences. Maximum-likelihood and Bayesian phylogenetic inferences showed two well-supported, reciprocally monophyletic lineages corresponding to POW and DTV. Bayesian skyline plots based on year-of-sampling data indicated no significant population size change for either virus lineage. Statistical model-based selection analyses showed evidence of purifying selection in both lineages. Positive selection was detected in NS-5 sequences for both lineages and envelope sequences for POW. Our findings confirm that POW and DTV sequences are relatively stable over time, which suggests strong evolutionary constraint, and support field observations that suggest that tick-borne flavivirus populations are extremely stable in enzootic foci.
Analysis of Evolutionary Processes of Species Jump in Waterfowl Parvovirus

PubMed Central

Fan, Wentao; Sun, Zhaoyu; Shen, Tongtong; Xu, Danning; Huang, Kehe; Zhou, Jiyong; Song, Suquan; Yan, Liping

2017-01-01

Waterfowl parvoviruses are classified into goose parvovirus (GPV) and Muscovy duck parvovirus (MDPV) according to their antigenic features and host preferences. A novel duck parvovirus (NDPV), identified as a new variant of GPV, is currently infecting ducks, thus causing considerable economic loss. This study analyzed the molecular evolution and population dynamics of the emerging parvovirus capsid gene to investigate the evolutionary processes concerning the host shift of NDPV. Two important amino acids changes (Asn-489 and Asn-650) were identified in NDPV, which may be responsible for host shift of NDPV. Phylogenetic analysis indicated that the currently circulating NDPV originated from the GPV lineage. The Bayesian Markov chain Monte Carlo tree indicated that the NDPV diverged from GPV approximately 20 years ago. Evolutionary rate analyses demonstrated that GPV evolved with 7.674 × 10-4 substitutions/site/year, and the data for MDPV was 5.237 × 10-4 substitutions/site/year, whereas the substitution rate in NDPV branch was 2.25 × 10-3 substitutions/site/year. Meanwhile, viral population dynamics analysis revealed that the GPV major clade, including NDPV, grew exponentially at a rate of 1.717 year-1. Selection pressure analysis showed that most sites are subject to strong purifying selection and no positively selected sites were found in NDPV. The unique immune-epitopes in waterfowl parvovirus were also estimated, which may be helpful for the prediction of antibody binding sites against NDPV in ducks. PMID:28352261
No evidence for positive selection at two potential targets for malaria transmission-blocking vaccines in Anopheles gambiae s.s.

PubMed

Crawford, Jacob E; Rottschaefer, Susan M; Coulibaly, Boubacar; Sacko, Madjou; Niaré, Oumou; Riehle, Michelle M; Traore, Sékou F; Vernick, Kenneth D; Lazzaro, Brian P

2013-06-01

Human malaria causes nearly a million deaths in sub-Saharan Africa each year. The evolution of drug-resistance in the parasite and insecticide resistance in the mosquito vector has complicated control measures and made the need for new control strategies more urgent. Anopheles gambiae s.s. is one of the primary vectors of human malaria in Africa, and parasite-transmission-blocking vaccines targeting Anopheles proteins have been proposed as a possible strategy to control the spread of the disease. However, the success of these hypothetical technologies would depend on the successful ability to broadly target mosquito populations that may be genetically heterogeneous. Understanding the evolutionary pressures shaping genetic variation among candidate target molecules offers a first step towards evaluating the prospects of successfully deploying such technologies. We studied the population genetics of genes encoding two candidate target proteins, the salivary gland protein saglin and the basal lamina structural protein laminin, in wild populations of the M and S molecular forms of A. gambiae in Mali. Through analysis of intraspecific genetic variation and interspecific comparisons, we found no evidence of positive natural selection at the genes encoding these proteins. On the contrary, we found evidence for particularly strong purifying selection at the laminin gene. These results provide insight into the patterns of genetic diversity of saglin and laminin, and we discuss these findings in relation to the potential development of these molecules as vaccine targets. Copyright © 2013 Elsevier B.V. All rights reserved.
Trends of the Major Porin Gene (ompF) Evolution: Insight from the Genus Yersinia

PubMed Central

Stenkova, Anna M.; Isaeva, Marina P.; Shubin, Felix N.; Rasskazov, Valeri A.; Rakin, Alexander V.

2011-01-01

OmpF is one of the major general porins of Enterobacteriaceae that belongs to the first line of bacterial defense and interactions with the biotic as well as abiotic environments. Porins are surface exposed and their structures strongly reflect the history of multiple interactions with the environmental challenges. Unfortunately, little is known on diversity of porin genes of Enterobacteriaceae and the genus Yersinia especially. We analyzed the sequences of the ompF gene from 73 Yersinia strains covering 14 known species. The phylogenetic analysis placed most of the Yersinia strains in the same line assigned by 16S rDNA-gyrB tree. Very high congruence in the tree topologies was observed for Y. enterocolitica, Y. kristensenii, Y. ruckeri, indicating that intragenic recombination in these species had no effect on the ompF gene. A significant level of intra- and interspecies recombination was found for Y. aleksiciae, Y. intermedia and Y. mollaretii. Our analysis shows that the ompF gene of Yersinia has evolved with nonrandom mutational rate under purifying selection. However, several surface loops in the OmpF porin contain positively selected sites, which very likely reflect adaptive diversification Yersinia to their ecological niches. To our knowledge, this is a first investigation of diversity of the porin gene covering the whole genus of the family Enterobacteriaceae. This study demonstrates that recombination and positive selection both contribute to evolution of ompF, but the relative contribution of these evolutionary forces are different among Yersinia species. PMID:21655186
Quinazoline derivative from indigenous isolate, Nocardiopsis alba inhibits human telomerase enzyme.

PubMed

Kiran, K G; Thandeeswaran, M; Ayub Nawaz, K A; Easwaran, M; Jayagopi, K K; Ebrahimi, L; Palaniswamy, M; Mahendran, R; Angayarkanni, J

2016-12-01

Aim of this study was isolation and screening of various secondary metabolites produced by indigenous isolates of soil Actinomycetes for human telomerase inhibitory activity. Extracellular extract from culture suspension of various soil Actinomycetes species were tested for telomerase inhibitory activity. The organism which produced telomerase inhibitor was identified by 16S rRNA gene sequencing. The active fraction was purified by HPLC and analysed by GC-MS to identify the compound. In GC-MS analysis, the active principle was identified as 3-[4'-(2″-chlorophenyl)-2'-thiazolyl]-2,4-dioxo-1,2,3,4-tetrahydro quinazoline. The G-quadruplex stabilizing ability of the compound was checked by molecular docking and simulation experiments with G-quadruplex model (PDB ID-1L1H). The selective binding ability of the compound with G-quadruplex over Dickerson-Drew dodecamer DNA structures showed that the compound possess high selectivity towards G-quadruplex. Quinazoline derivative isolated from an indigenous strain of Nocardiopsis alba inhibited telomerase. Molecular docking and simulation studies predicted that this compound is a strong stabilizer of G-quadruplex conformation. It also showed a preferable binding to G-quadruplex DNA over normal DNA duplex. This particular compound can be suggested as a suitable compound for developing a future anticancer drug. The selectivity towards G-quadruplex over normal DNA duplex gives a clue that it is likely to show lower cytotoxicity in normal cells. © 2016 The Society for Applied Microbiology.
Crude and purified proteasome activity assays are affected by type of microplate.

PubMed

Cui, Ziyou; Gilda, Jennifer E; Gomes, Aldrin V

2014-02-01

Measurement of proteasome activity is fast becoming a commonly used assay in many laboratories. The most common method to measure proteasome activity involves measuring the release of fluorescent tags from peptide substrates in black microplates. Comparisons of black plates used for measuring fluorescence with different properties show that the microplate properties significantly affect the measured activities of the proteasome. The microplate that gave the highest reading of trypsin-like activity of the purified 20S proteasome gave the lowest reading of chymotrypsin-like activity of the 20S proteasome. Plates with medium binding surfaces from two different companies showed an approximately 2-fold difference in caspase-like activity for purified 20S proteasomes. Even standard curves generated using free 7-amino-4-methylcoumarin (AMC) were affected by the microplate used. As such, significantly different proteasome activities, as measured in nmol AMC released/mg/min, were obtained for purified 20S proteasomes as well as crude heart and liver samples when using different microplates. The naturally occurring molecule betulinic acid activated the chymotrypsin-like proteasome activity in three different plates but did not affect the proteasome activity in the nonbinding surface microplate. These findings suggest that the type of proteasome activity being measured and sample type are important when selecting a microplate. Copyright © 2013 Elsevier Inc. All rights reserved.
Growth inhibitory and proapoptotic effects of l-asparaginase from Fusarium culmorum ASP-87 on human leukemia cells (Jurkat).

PubMed

Meghavarnam, Anil K; Salah, Maryam; Sreepriya, Meenakshisundaram; Janakiraman, Savitha

2017-06-01

The objective of this study was to evaluate the anticancer properties of l-asparaginase purified from fungal isolate Fusarium culmorum ASP-87 against human T-cell leukemia cell line (Jurkat). The growth inhibitory and proapoptotic effects of purified l-asparaginase on Jurkat cell lines were investigated by determining its influence on cell viability, colony formation, DNA fragmentation, and cell cycle progression. The results revealed that purified l-asparaginase showed significant decrease in cell survival with IC 50 value of 90 μg/mL (9 IU/mL). The enzyme inhibited colony formation and showed characteristic laddering pattern on agarose gel thereby confirming the induction of apoptosis. Further, cell cycle analysis revealed that the enzyme induced apoptotic cell death by arresting the growth of cells at G 2 -M phase. However, the enzyme did not elicit any toxic effects on human erythrocytes. l-asparaginase purified from F. culmorum ASP-87 showed significant and selective cytotoxic and apoptotic effects on human T-cell leukemic cells in dose-dependent manner. Results of the study give leads for the anticancer effects of fungal l-asparaginase and its potential usefulness in the chemotherapy of leukemia. © 2016 Société Française de Pharmacologie et de Thérapeutique.

[Cloning, expression and identification of functional fragment rC3B of human complement C3 in E. Coli].

PubMed

Gan, Hui; Zhou, Yong; Sun, Ping; Zhu, Xiao-Xia; Wang, Quan-Li; Zhan, Lin-Sheng

2007-08-01

This study was purposed to verify the binding part of human complement C3 to complement receptor III (CRIII) in monocytes, the peptide rC3B, including the binding-site, was expressed, purified and identified. rC3B, the binding part of human complement C3 to CRIII, was selected by computer-aided modeling and summarizing researches published. Then, rC3B gene fragment was amplified by PCR, and cloned into prokaryotic vector pQE30a. The fusion protein rC3B was expressed in E.coli M15 and purified by Ni(2+)-chelating affinity chromatography. The activity of rC3B was identified by Western blot and adherence assay with monocytes. The results showed that rC3B fragment was obtained, and a prokaryotic expression vector pQE30-rC3B was constructed. rC3B was efficiently expressed and purified. In Western blot, the target protein showed the activity of binding with C3 antibody, while the purified protein showed the activity of adherence with monocytes. It is concluded that the recombinant C3B was obtained and identified, and this study lay the basis for the further functional analysis of C3.
Photocatalytic air purifiers for indoor air: European standard and pilot room experiments.

PubMed

Costarramone, N; Cantau, C; Desauziers, V; Pécheyran, C; Pigot, T; Lacombe, S

2017-05-01

At the European level (CEN/TC386), some efforts are currently devoted to new standards for comparing the efficiency of commercial photocatalytic material/devices in various application fields. Concerning prototype or commercial indoor photocatalytic air purifiers designed for volatile organic compounds (VOC) abatement, the methodology is based on a laboratory airtight chamber. The photocatalytic function is demonstrated by the mineralization of a mixture of five VOCs. Experimental data were obtained for four selected commercial devices and three commercial materials: drop of VOC concentration, but also identification of secondary species (with special attention to formaldehyde), mineralization rates, and Clean Air Delivery Rate (CADR). With two efficient air purifiers, these laboratory experiments were compared to the results in two experimental rooms (35-40 m 3 ) where air pollution was introduced through wooden floor and furniture. The systems' ageing was also studied. The safety of the commercial products was also assessed by the determination of nanoparticle release. Standardized tests are useful to rank photocatalytic air purifiers and passive materials and to discard inefficient ones. A good correlation between the standard experiments and the experimental room experiments was found, even if in the latter case, the concentration of lower weight VOCs drops less quickly than that of heavier VOCs.
Overproduction and partial purification of the Norrie disease gene product, norrin, from a recombinant baculovirus.

PubMed

Shastry, Barkur S; Trese, Michael T

2003-12-05

Abnormal vascularization of the peripheral retina and retinal detachment are common clinical characteristics of Norrie disease (ND), familial exudative vitreoretinopathy, Coats' disease, and retinopathy of prematurity. Although little is known about the molecular basis of these diseases, studies have shown that all of these diseases are associated with mutations in the ND gene. In spite of this, little is known about norrin, its molecular mechanism of action, and its functional relationship with the development of abnormal retinal vasculature. To obtain a large quantity of norrin for structural and functional studies, we have overproduced it in insect cells. For this purpose, a cDNA fragment (869 bp) was isolated from a human retinal cDNA library by amplification and was cloned into an expression vector. The purified plasmid was co-transfected with wild-type linearized Bac-N-Blue DNA into S. frugiperda Sf21 insect cells. The recombinant virus plaques were purified and clones were selected based on the level of recombinant protein expressed in Sf21 cells infected with a purified recombinant virus. From these, a high-titer stock was generated and subsequently used to prepare a fused protein on a large scale. The protein was partially purified by the process of immobilized metal affinity chromatography and the use of ion exchange chromatography
Structure and evolution analysis of pollen receptor-like kinase in Zea mays and Arabidopsis thaliana.

PubMed

Wang, Dongxu; Wang, He; Irfan, Muhammad; Fan, Mingxia; Lin, Feng

2014-08-01

Receptor-like kinase (RLKs) is an important member in protein kinase family which is widely involved in plant growth, development and defense responses. It is significant to analyze the kinase structure and evolution of pollen RLKs in order to study their mechanisms. In our study, 64 and 73 putative pollen RLKs were chosen from maize and Arabidopsis. Phylogenetic analysis showed that the pollen RLKs were conservative and might had existed before divergence between monocot and dicot which were mainly concentrated in RLCK-VII and LRR-III two subfamilies. Chromosomal localization and gene duplication analysis showed the expansion of pollen RLKs were mainly caused by segmental duplication. By calculating Ka/Ks value of extracellular domain, intracellular domain and kinase domain in pollen RLKs, we found that the pollen RLKs duplicated genes had mainly experienced the purifying selection, while maize might have experienced weaker purifying selection. Meanwhile, extracellular domain might have experienced stronger diversifying selection than intracellular domain in both species. Estimation of duplication time showed that the duplication events of Arabidopsis have occurred approximately between 18 and 69 million years ago, compared to 0.67-170 million years ago of maize. Copyright © 2014 Elsevier Ltd. All rights reserved.
Purification and characterization of a casein kinase 2-type protein kinase from pea nuclei

NASA Technical Reports Server (NTRS)

Li, H.; Roux, S. J.

1992-01-01

Almost all the polyamine-stimulated protein kinase activity associated with the chromatin fraction of nuclei purified from etiolated pea (Pisum sativum L.) plumules is present in a single enzyme that can be extracted from chromatin by 0.35 molar NaCl. This protein kinase can be further purified over 2000-fold by salt fractionation and anion-exchange and casein-agarose column chromatography, after which it is more than 90% pure. The purified kinase has a specific activity of about 650 nanomoles per minute per milligram protein in the absence of polyamines, with either ATP or GTP as phosphoryl donor. Spermidine can stimulate its activity fourfold, with half-maximal activation at about 2 millimolar. Spermine and putrescine also stimulate activity, although somewhat less effectively. This kinase has a tetrameric alpha 2 beta 2 structure with a native molecular weight of 130,000, and subunit molecular weights of 36,000 for the catalytic subunit (alpha) and 29,000 for the regulatory subunit (beta). In western blot analyses, only the alpha subunit reacts strongly with polyclonal antibodies to a Drosophila casein kinase II. The pea kinase can use casein and phosvitin as artificial substrates, phosphorylating both the serine and threonine residues of casein. It has a pH optimum near 8.0, a Vmax of 1.5 micromoles per minute per milligram protein, and a Km for ATP of approximately 75 micromolar. Its activity can be almost completely inhibited by heparin at 5 micrograms per milliliter, but is relatively insensitive to concentrations of staurosporine, K252a, and chlorpromazine that strongly antagonize Ca(2+) -regulated protein kinases. These results are discussed in relation to recent findings that casein kinase 2-type kinases may phosphorylate trans-acting factors that bind to light-regulated promoters in plants.
Anti-methicillin-resistance Staphylococcus aureus (MRSA) compounds from Bauhinia kockiana Korth. And their mechanism of antibacterial activity.

PubMed

Chew, Yik Ling; Mahadi, Adlina Maisarah; Wong, Kak Ming; Goh, Joo Kheng

2018-02-20

Bauhinia kockiana originates from Peninsular Malaysia and it is grown as a garden ornamental plant. Our previous study reported that this plant exhibited fairly strong antioxidant and antimicrobial activities. This paper focused on the assessment of the antibacterial activity of B. kockiana towards methicillin-resistance Staphylococcus aureus (MRSA), to purify and to identify the antibacterial compounds, and to determine the mechanism of antibacterial activity. Antibacterial activity of B. kockiana flower was evaluated qualitatively and quantitatively using disc diffusion assay and microbroth dilution method. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of extracts were examined. Phytochemical analysis was performed to determine the classes of phytochemicals in the extracts. Bioactivity guided isolation was employed to purify the antibacterial agents and identified via various spectroscopy methods. Scanning electron microscopy (SEM) technique was used to evaluate the antibacterial mechanism of extract and compounds isolated. B. kockiana flower was found to exhibit fairly strong antibacterial activity towards both strains of MRSA bacteria used, MIC varies from 62.5-250 μg/mL. Tannins and flavonoids have been detected in the phytochemical analysis. Gallic acid and its ester derivatives purified from ethyl acetate extract could inhibit MRSA at 250-500 μg/mL. SEM revealed that the cells have undergone plasmolysis upon treatment with the extract and compounds. Tannins and polyphenols are the antibacterial components towards MRSA in B. kockiana. Massive leakage of the cell content observed in treated cells showed that the phytochemicals have changed the properties of the cell membranes. Amphiphilic nature of the compounds exhibited the antibacterial activity towards MRSA via three stages: (1) cell membrane attachment; (2) cell membrane fluidity modification; and (3) cell membrane structure disruption.
Identification and characterization of α1 -antitrypsin in fibrin clots.

PubMed

Talens, S; Malfliet, J J M C; van Hal, P Th W; Leebeek, F W G; Rijken, D C

2013-07-01

Preliminary studies indicated that α1 -antitrypsin (A1AT) is the most abundant protein that is non-covalently bound to fibrin clots prepared from plasma. The aim of this study was to identify and characterize fibrin(ogen)-bound A1AT. Plasma clots were prepared and extensively washed with saline. Clot-bound A1AT could only be extracted using denaturing agents such as urea, thiourea or SDS, pointing to an apparently strong association. Purified fibrinogen, but still containing A1AT as a contaminant, was gel filtered, which showed that the A1AT was bound to fibrinogen. A specific ELISA detected the presence of A1AT-fibrinogen complexes in both purified fibrinogen and pooled normal plasma. Finally, fibrin(ogen)-Sepharose chromatography indicated that A1AT purified from plasma contained a small fraction of fibrin(ogen)-binding A1AT. To study the inhibitory activity of fibrin(ogen)-bound A1AT, both fibrinogen containing A1AT and washed plasma clots were incubated with increasing amounts of elastase. SDS-PAGE and Western blotting showed under both conditions the generation of the A1AT-elastase complex as well as cleaved A1AT. The inhibitory activity of fibrin(ogen)-bound A1AT was also demonstrated by measuring elastase-induced lysis of fibrin clots. Fibrin clots contain strongly bound A1AT, which is functionally active as a serine protease inhibitor (serpin). This A1AT might play a role in the local regulation of proteases involved in coagulation or fibrinolysis and represent a novel link between the inflammatory and hemostatic systems. © 2013 International Society on Thrombosis and Haemostasis.
Enzymatic characterization of transglutaminase from Streptomyces mobaraensis DSM 40587 in high salt and effect of enzymatic cross-linking of yak milk proteins on functional properties of stirred yogurt.

PubMed

Zhang, L; Zhang, L; Yi, H; Du, M; Ma, C; Han, X; Feng, Z; Jiao, Y; Zhang, Y

2012-07-01

Streptomyces transglutaminase (TGase) purified from high-salt medium was characterized and applied into yak yogurts. The purified enzyme presented a Michaelis constant of 40.47 mmol and a maximum velocity of 44.44 U/mg of protein for N-carboxybenzoyl-l-glutaminyl-glycine in the hydroxamate procedure. The purified TGase exhibited optimum activity at 55°C and pH 6.0. The enzyme was not stable above 50°C and was stable within a pH range of 5.0 to 10.0 at 4°C for 12h and pH 5.0 to 9.0 at 37°C for 30 min. The TGase activity was not affected by Ca(2+), K(+), Ba(2+), or Na(+), but slightly inhibited by Fe(2+), Mg(2+), and Mn(2+), and strongly by Cu(2+) and Zn(2+). To explore yak milk products, it was used to produce yogurt and TGase was used. It was found that TGase-catalyzed cross-linking was effective in improving functional properties of stirred yak yogurt. Treated yogurt produced a strong acid gel, higher consistency, cohesiveness, index of viscosity, and creamier mouth feel than the untreated product. Furthermore, yak yogurt treated with TGase presented lower wet yak hair or sweat odor, or both. Therefore, TGase can be used to pave the way for exploration of novel yak products to overcome the issues of peculiar wet yak hair or sweat odor, or both. Copyright © 2012 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.
Calcium Channels: Structure and Function (Annals of the New York Academy of Sciences. Volume 560)

DTIC Science & Technology

1989-06-26

many protease inhibitors were used , we believe that proteolysis was a problem. We therefore modified our purification protocol and have obtained a...recover activity by selective combination of fractions were unsuccessful. Chemical Cross-Linking of [ 25 Jo-CgTX Cross-linking of w-CgTX derivatives using ... using the planar bilayer recording technique and by comparing ligand-dependent gating, ionic selectivity , and pharmacology of purified ryanodine
Preparation of stable isotope-labeled peripheral cannabinoid receptor CB2 by bacterial fermentation

PubMed Central

Berger, Christian; Ho, Jenny T.C.; Kimura, Tomohiro; Hess, Sonja; Gawrisch, Klaus; Yeliseev, Alexei

2010-01-01

We developed a bacterial fermentation protocol for production of a stable isotope-labeled cannabinoid receptor CB2 for subsequent structural studies of this protein by nuclear magnetic resonance spectroscopy. The human peripheral cannabinoid receptor was expressed in Escherichia coli as a fusion with maltose binding protein and two affinity tags. The fermentation was performed in defined media comprised of mineral salts, glucose and 15N2-L-tryptophan to afford incorporation of the labeled amino acid into the protein. Medium, growth and expression conditions were optimized so that the fermentation process produced about 2 mg of purified, labeled CB2 per liter of culture medium. By performing a mass spectroscopic characterization of the purified CB2, we determined that one of the two 15N atoms in tryptophan was incorporated into the recombinant protein. NMR analysis of 15N chemical shifts strongly suggests that the 15N atoms are located in Trp-indole rings. Importantly, analysis of the peptides derived from the CNBr cleavage of the purified protein confirmed a minimum of 95% incorporation of the labeled tryptophan into the CB2 sequence. The labeled CB2, purified and reconstituted into liposomes at a protein-to-lipid molar ratio of 1:500, was functional as confirmed by activation of cognate G proteins in an in vitro coupled assay. To our knowledge, this is the first reported production of a biologically active, stable isotope-labeled G protein-coupled receptor by bacterial fermentation. PMID:20044006
A Bowman-Birk protease inhibitor purified, cloned, sequenced and characterized from the seeds of Maclura pomifera (Raf.) Schneid.

PubMed

Indarte, Martín; Lazza, Cristian M; Assis, Diego; Caffini, Néstor O; Juliano, María A; Avilés, Francesc X; Daura, Xavier; López, Laura M I; Trejo, Sebastián A

2017-02-01

A new BBI-type protease inhibitor with remarkable structural characteristics was purified, cloned, and sequenced from seeds of Maclura pomifera , a dicotyledonous plant belonging to the Moraceae family. In this work, we report a Bowman-Birk inhibitor (BBI) isolated, purified, cloned, and characterized from Maclura pomifera seeds (MpBBI), the first of this type from a species belonging to Moraceae family. MpBBI was purified to homogeneity by RP-HPLC, total RNA was extracted from seeds of M. pomifera, and the 3'RACE-PCR method was applied to obtain the cDNA, which was cloned and sequenced. Peptide mass fingerprinting (PMF) analysis showed correspondence between the in silico-translated protein and MpBBI, confirming that it corresponds to a new plant protease inhibitor. The obtained cDNA encoded a polypeptide of 65 residues and possesses 10 cysteine residues, with molecular mass of 7379.27, pI 6.10, and extinction molar coefficient of 9105 M -1 cm -1 . MpBBI inhibits strongly trypsin with K i in the 10 -10 M range and was stable in a wide array of pH and extreme temperatures. MpBBI comparative modeling was applied to gain insight into its 3D structure and highlighted some distinguishing features: (1) two non-identical loops, (2) loop 1 (CEEESRC) is completely different from any known BBI, and (3) the amount of disulphide bonds is also different from any reported BBI from dicot plants.
Recombinant dengue 2 virus NS3 protein conserves structural antigenic and immunological properties relevant for dengue vaccine design.

PubMed

Ramírez, Rosa; Falcón, Rosabel; Izquierdo, Alienys; García, Angélica; Alvarez, Mayling; Pérez, Ana Beatriz; Soto, Yudira; Muné, Mayra; da Silva, Emiliana Mandarano; Ortega, Oney; Mohana-Borges, Ronaldo; Guzmán, María G

2014-10-01

The NS3 protein is a multifunctional non-structural protein of flaviviruses implicated in the polyprotein processing. The predominance of cytotoxic T cell lymphocytes epitopes on the NS3 protein suggests a protective role of this protein in limiting virus replication. In this work, we studied the antigenicity and immunogenicity of a recombinant NS3 protein of the Dengue virus 2. The full-length NS3 gene was cloned and expressed as a His-tagged fusion protein in Escherichia coli. The pNS3 protein was purified by two chromatography steps. The recombinant NS3 protein was recognized by anti-protease NS3 polyclonal antibody and anti-DENV2 HMAF by Western Blot. This purified protein was able to stimulate the secretion of high levels of gamma interferon and low levels of interleukin-10 and tumor necrosis factor-α in mice splenocytes, suggesting a predominantly Th-1-type T cell response. Immunized BALB/c mice with the purified NS3 protein showed a strong induction of anti-NS3 IgG antibodies, essentially IgG2b, as determined by ELISA. Immunized mice sera with recombinant NS3 protein showed specific recognition of native dengue protein by Western blotting and immunofluorescence techniques. The successfully purified recombinant protein was able to preserv the structural and antigenic determinants of the native dengue protein. The antigenicity shown by the recombinant NS3 protein suggests its possible inclusion into future DENV vaccine preparations.
Neurotrophic requirements of human motor neurons defined using amplified and purified stem cell-derived cultures.

PubMed

Lamas, Nuno Jorge; Johnson-Kerner, Bethany; Roybon, Laurent; Kim, Yoon A; Garcia-Diaz, Alejandro; Wichterle, Hynek; Henderson, Christopher E

2014-01-01

Human motor neurons derived from embryonic and induced pluripotent stem cells (hESCs and hiPSCs) are a potentially important tool for studying motor neuron survival and pathological cell death. However, their basic survival requirements remain poorly characterized. Here, we sought to optimize a robust survival assay and characterize their response to different neurotrophic factors. First, to increase motor neuron yield, we screened a small-molecule collection and found that the Rho-associated kinase (ROCK) inhibitor Y-27632 enhances motor neuron progenitor proliferation up to 4-fold in hESC and hiPSC cultures. Next, we FACS-purified motor neurons expressing the Hb9::GFP reporter from Y-27632-amplified embryoid bodies and cultured them in the presence of mitotic inhibitors to eliminate dividing progenitors. Survival of these purified motor neurons in the absence of any other cell type was strongly dependent on neurotrophic support. GDNF, BDNF and CNTF all showed potent survival effects (EC(50) 1-2 pM). The number of surviving motor neurons was further enhanced in the presence of forskolin and IBMX, agents that increase endogenous cAMP levels. As a demonstration of the ability of the assay to detect novel neurotrophic agents, Y-27632 itself was found to support human motor neuron survival. Thus, purified human stem cell-derived motor neurons show survival requirements similar to those of primary rodent motor neurons and can be used for rigorous cell-based screening.
Neurotrophic Requirements of Human Motor Neurons Defined Using Amplified and Purified Stem Cell-Derived Cultures

PubMed Central

Lamas, Nuno Jorge; Johnson-Kerner, Bethany; Roybon, Laurent; Kim, Yoon A.; Garcia-Diaz, Alejandro; Wichterle, Hynek; Henderson, Christopher E.

2014-01-01

Human motor neurons derived from embryonic and induced pluripotent stem cells (hESCs and hiPSCs) are a potentially important tool for studying motor neuron survival and pathological cell death. However, their basic survival requirements remain poorly characterized. Here, we sought to optimize a robust survival assay and characterize their response to different neurotrophic factors. First, to increase motor neuron yield, we screened a small-molecule collection and found that the Rho-associated kinase (ROCK) inhibitor Y-27632 enhances motor neuron progenitor proliferation up to 4-fold in hESC and hiPSC cultures. Next, we FACS-purified motor neurons expressing the Hb9::GFP reporter from Y-27632-amplified embryoid bodies and cultured them in the presence of mitotic inhibitors to eliminate dividing progenitors. Survival of these purified motor neurons in the absence of any other cell type was strongly dependent on neurotrophic support. GDNF, BDNF and CNTF all showed potent survival effects (EC50 1–2 pM). The number of surviving motor neurons was further enhanced in the presence of forskolin and IBMX, agents that increase endogenous cAMP levels. As a demonstration of the ability of the assay to detect novel neurotrophic agents, Y-27632 itself was found to support human motor neuron survival. Thus, purified human stem cell-derived motor neurons show survival requirements similar to those of primary rodent motor neurons and can be used for rigorous cell-based screening. PMID:25337699
Characterization of endo-β-mannanase from Enterobacter ludwigii MY271 and application in pulp industry.

PubMed

Yang, Miao; Cai, Jun; Wang, Changgao; Du, Xin; Lin, Jianguo

2017-01-01

β-Mannanases are the second most important enzymes for the hydrolysis of hemicelluloses. An endo-β-mannanase from Enterobacter ludwigii MY271 was purified at 11.7 ± 0.2-fold to homogeneity with a final recovery of 15.2 ± 0.2 %. Using purified β-mannanase protein and SDS-PAGE, the molecular mass was found to be 43.16 kDa. The optimal pH and temperature of the enzyme was found to be 7.0 and 55 °C, respectively. The β-mannanase activity was stable over a broad pH range of pH 2.0-10.0. In addition, the purified enzyme was highly activated by several metal ions and chemical reagents, such as Mg 2+ , L-cysteine, glutathione (GSH) and β-mercaptoethanol. Whereas the enzyme was strongly inhibited by Hg 2+ , Cu 2+ , N-bromosuccinimide (NBS), 1-ethyl-3-(3-dimethyl-amino-propyl)-carbodiimide (EDC), phenylmethanesulfonyl fluoride (PMSF), and sodium dodecyl sulfate (SDS). The β-mannanase was highly active towards glucomannan, and showed endo-activity by producing a mixture of oligosaccharides. Moreover, the enzyme displayed a classical endo-type mode on mannooligosaccharides. The β-mannanase coupled with xylanase significantly improved the brightness of kraft pulp, whereas it has no remarkable effect on the tensile strength of the pulp. Our functional studies of the purified β-mannanase indicate that the enzyme is beneficial to industrial applications, in particular, biotechnological processes, such as food, feed and pulp industry.
Selective Activation of Transcription by a Novel CCAAT Binding Factor

NASA Astrophysics Data System (ADS)

Maity, Sankar N.; Golumbek, Paul T.; Karsenty, Gerard; de Crombrugghe, Benoit

1988-07-01

A novel CCAAT binding factor (CBF) composed of two different subunits has been extensively purified from rat liver. Both subunits are needed for specific binding to DNA. Addition of this purified protein to nuclear extracts of NIH 3T3 fibroblasts stimulates transcription from several promoters including the α 2(I) collagen, the α 1(I) collagen, the Rous sarcoma virus long terminal repeat (RSV-LTR), and the adenovirus major late promoter. Point mutations in the CCAAT motif that show either no binding or a decreased binding of CBF likewise abolish or reduce activation of transcription by CBF. Activation of transcription requires, therefore, the specific binding of CBF to its recognition sites.
Comparison of the amino acid compositions and antigenic properties of spiralins purified from the plasma membranes of different spiroplasmas.

PubMed

Wróblewski, H; Robic, D; Thomas, D; Blanchard, A

1984-01-01

Spiralins were purified by agarose-suspension electrophoresis after extraction with detergents from the membranes of the following spiroplasmas: Spiroplasma citri C189, S. citri Maroc (R8A2), S. citri Scaph and the honey-bee spiroplasma B88. The four proteins (molecular mass congruent to 26,000 daltons, as determined by sodium dodecyl sulphate-pore gradient electrophoresis) showed very similar amino acid compositions characterized by the absence of methionine and tryptophan and a high polarity index (greater than 49%). When compared with the amino acid composition of S. citri membrane, the four spiralins had little or no histidine, a low content of glycine, leucine, tyrosine, phenylalanine and arginine, and a high content of threonine, alanine and valine. Comparison of the amino acid compositions according to the criteria described by Cornish-Bowden (Anal. Biochem., 1980, 105, 233-238) strongly suggests that all four spiralins are related. A crossed immunoelectrophoretical comparison, however, shows that though the three proteins purified from S. citri strains (serogroup I-1) are antigenically similar, they do not seem to share common epitopes with spiralin from the honey-bee spiroplasma B88 (serogroup I-2).
Multiple forms of ADP-glucose pyrophosphorylase from tomato fruit

NASA Technical Reports Server (NTRS)

Chen, B. Y.; Janes, H. W.

1997-01-01

ADP-glucose pyrophosphorylase (AGP) was purified from tomato (Lycopersicon esculentum Mill.) fruit to apparent homogeneity. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis the enzyme migrated as two close bands with molecular weights of 50,000 and 51,000. Two-dimensional polyacrylamide gel electrophoresis analysis of the purified enzyme, however, revealed at least five major protein spots that could be distinguished by their slight differences in net charge and molecular weight. Whereas all of the spots were recognized by the antiserum raised against tomato fruit AGP holoenzyme, only three of them reacted strongly with antiserum raised against the potato tuber AGP large subunit, and the other two spots (with lower molecular weights) reacted specifically with antisera raised against spinach leaf AGP holoenzyme and the potato tuber AGP small subunit. The results suggest the existence of at least three isoforms of the AGP large subunit and two isoforms of the small subunit in tomato fruit in vivo. The native molecular mass of the enzyme determined by gel filtration was 220 +/- 10 kD, indicating a tetrameric structure for AGP from tomato fruit. The purified enzyme is very sensitive to 3-phosphoglycerate/inorganic phosphate regulation.
Extraction, purification and elicitor activities of polysaccharides from Chrysanthemum indicum.

PubMed

Du, Ningning; Tian, Wei; Zheng, Dongfang; Zhang, Xinyi; Qin, Pinyan

2016-01-01

Polysaccharides isolated from Chrysanthemum indicum were studied for their pathogen-derived resistance against Sclerotium rolfsii sacc in Atractylodis maceocephalae koidz. The total sugar content and monosaccharide analysis were determined by phenol-sulfuric acid method and gas chromatography, and infrared spectroscopy performed for simple structure information. The activities of CAT and POD as protective enzymes in A. maceocephalae leaves were evaluated. The purified polysaccharides exhibited strong CAT and POD activities in inoculated with S. rolfsii in A. macrocephala leaves, attained the maximum value 568.3 Ug(-1)min(-1) and 604.4 Ug(-1)min(-1)respectively. Whereas, when compared with the control plants, 20mg/ml purified polysaccharides exhibited the strongest CAT and POD activities. Notably, the treatments of A. macepcephalae seedlings with C. indicum polysaccharides (CIP) decreased disease index development caused by S. rolfsii. The disease index after 10 days was significantly reduced when the seedlings treated with 20mg/ml CIP, 4.41 compared to the control plants 32.00. Given together, these results indicated that purified polysaccharides derived from C. indicum may be useful as a natural inducer. Copyright © 2015 Elsevier B.V. All rights reserved.
Purification and characterization of nattokinase from Bacillus subtilis natto B-12.

PubMed

Wang, Cong; Du, Ming; Zheng, Dongmei; Kong, Fandong; Zu, Guoren; Feng, Yibing

2009-10-28

Bacillus subtilis natto B-12 was isolated from natto, a traditional fermented soybean food in Japan. A fibrinolytic enzyme (B-12 nattokinase) was purified from the supernatant of B. subtilis natto B-12 culture broth and showed strong fibrinolytic activity. The enzyme was homogenously purified to 56.1-fold, with a recovery of 43.2% of the initial activity. B-12 nattokinase was demonstrated to be homogeneous by SDS-PAGE and was identified as a monomer of 29000 +/- 300 Da in its native state by SDS-PAGE and size exclusion methods. The optimal pH value and temperature were 8.0 and 40 degrees C, respectively. Purified nattokinase showed high thermostability at temperatures from 30 to 50 degrees C and alkaline stability within the range of pH 6.0-9.0. The enzyme activity was activated by Zn(2+) and obviously inhibited by Fe(3+) and Al(3+). This study provides some important information for the effect factors of fibrinolytic activity, the purification methods, and characterization of nattokinase from B. subtilis natto B-12, which enriches the theoretical information of nattokinase for the research and development of nattokinase as a functional additive of food.

Flavonoids inhibit both rice and sheep serotonin N-acetyltransferases and reduce melatonin levels in plants.

PubMed

Lee, Kyungjin; Hwang, Ok Jin; Reiter, Russel J; Back, Kyoungwhan

2018-05-31

The plant melatonin biosynthetic pathway has been well characterized, but inhibitors of melatonin synthesis have not been well studied. Here, we found that flavonoids potently inhibited plant melatonin synthesis. For example, flavonoids including morin and myricetin significantly inhibited purified, recombinant sheep serotonin N-acetyltransferase (SNAT). Flavonoids also dose-dependently and potently inhibited purified rice SNAT1 and SNAT2. Thus, myricetin (100 μmol/L) reduced rice SNAT1 and SNAT2 activity 7- and 10-fold, respectively, and also strongly inhibited the N-acetylserotonin methyltransferase activity of purified, recombinant rice caffeic acid O-methyltransferase. To explore the in vivo effects, rice leaves were treated with flavonoids and then cadmium. Flavonoid-treated leaves had lower melatonin levels than the untreated control. To explore the direct roles of flavonoids in melatonin biosynthesis, we first functionally characterized a putative rice flavonol synthase (FLS) in vitro and generated flavonoid-rich transgenic rice plants that overexpressed FLS. Such plants produced more flavonoids but less melatonin than the wild-type, which suggests that flavonoids indeed inhibit plant melatonin biosynthesis. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.
Positive and purifying selection in mitochondrial genomes of a bird with mitonuclear discordance.

PubMed

Morales, Hernán E; Pavlova, Alexandra; Joseph, Leo; Sunnucks, Paul

2015-06-01

Diversifying selection on metabolic pathways can reduce intraspecific gene flow and promote population divergence. An opportunity to explore this arises from mitonuclear discordance observed in an Australian bird Eopsaltria australis. Across >1500 km, nuclear differentiation is low and latitudinally structured by isolation by distance, whereas two highly divergent, parapatric mitochondrial lineages (>6.6% in ND2) show a discordant longitudinal geographic pattern and experience different climates. Vicariance, incomplete lineage sorting and sex-biased dispersal were shown earlier to be unlikely drivers of the mitonuclear discordance; instead, natural selection on a female-linked trait was the preferred hypothesis. Accordingly, here we tested for signals of positive, divergent selection on mitochondrial genes in E. australis. We used codon models and physicochemical profiles of amino acid replacements to analyse complete mitochondrial genomes of the two mitochondrial lineages in E. australis, its sister species Eopsaltria griseogularis, and outgroups. We found evidence of positive selection on at least five amino acids, encoded by genes of two oxidative phosphorylation pathway complexes NADH dehydrogenase (ND4 and ND4L) and cytochrome bc1 (cyt-b) against a background of widespread purifying selection on all mitochondrial genes. Three of these amino acid replacements were fixed in ND4 of the geographically most widespread E. australis lineage. The other two replacements were fixed in ND4L and cyt-b of the geographically more restricted E. australis lineage. We discuss whether this selection may reflect local environmental adaptation, a by-product of other selective processes, or genetic incompatibilities, and propose how these hypotheses can be tested in future. © 2015 John Wiley & Sons Ltd.
40 CFR 1065.307 - Linearity verification.

Code of Federal Regulations, 2011 CFR

2011-07-01

... different flow rates. Use a gravimetric reference measurement (such as a scale, balance, or mass comparator... the gas-division system to divide the span gas with purified air or nitrogen. Select gas divisions... verification for gravimetric PM balances, use external calibration weights that that meet the requirements in...
Discovery of Culex pipiens associated tunisia virus: a new ssRNA(+) virus representing a new insect associated virus family

PubMed Central

Bigot, Diane; Atyame, Célestine M; Weill, Mylène; Justy, Fabienne

2018-01-01

Abstract In the global context of arboviral emergence, deep sequencing unlocks the discovery of new mosquito-borne viruses. Mosquitoes of the species Culex pipiens, C. torrentium, and C. hortensis were sampled from 22 locations worldwide for transcriptomic analyses. A virus discovery pipeline was used to analyze the dataset of 0.7 billion reads comprising 22 individual transcriptomes. Two closely related 6.8 kb viral genomes were identified in C. pipiens and named as Culex pipiens associated tunisia virus (CpATV) strains Ayed and Jedaida. The CpATV genome contained four ORFs. ORF1 possessed helicase and RNA-dependent RNA polymerase (RdRp) domains related to new viral sequences recently found mainly in dipterans. ORF2 and 4 contained a capsid protein domain showing strong homology with Virgaviridae plant viruses. ORF3 displayed similarities with eukaryotic Rhoptry domain and a merozoite surface protein (MSP7) domain only found in mosquito-transmitted Plasmodium, suggesting possible interactions between CpATV and vertebrate cells. Estimation of a strong purifying selection exerted on each ORFs and the presence of a polymorphism maintained in the coding region of ORF3 suggested that both CpATV sequences are genuine functional viruses. CpATV is part of an entirely new and highly diversified group of viruses recently found in insects, and that bears the genomic hallmarks of a new viral family. PMID:29340209
A Novel Marker for Purkinje Cells, Ribosomal Protein MPS1/S27: Expression of MPS1 in Human Cerebellum.

PubMed

Fernandez-Pol, J Alberto

2016-01-01

The ribosomal protein metallopanstimulin-1 (MPS1/S27) serves critical survival purposes in cell division, in normal and cancerous cells; for this reason, selective pressures of evolution have conserved the DNA sequences encoding MPS1/S27 in Archaea and Eukariotic cells. The expression of MPS1/S27 protein in human adult cerebellum has not been established. The presence of MPS1/S27, was screened in paraffin-embedded human adult brain specimens processed for tissue inmunohistochemistry. Affinity-purified specific antibodies were directed against the N-terminus of MPS1. The antibodies to MPS1 detected Purkinje cells (PC) and their dendrites. In PC, MPS1 antigen-positive staining was found in: the nucleolus, which was strongly stained; ribosomes attached to the external nuclear membrane; cytoplasm of PC, with strong staining in a punctuate fashion; the soma-attached large dendrite trunks of PC, which were MPS1 antigen-positive; and the granular cell layer, where cellular staining in a few cells that appeared to resemble smaller PC was observed. Since MPS1 is involved in cell division, DNA repair, and ribosomal biogenesis, it may be a useful antigen for studying processes such as protein synthesis, oncogenesis, regeneration, aging, and perhaps diseases of the human cerebellum. Copyright© 2016, International Institute of Anticancer Research (Dr. John G. Delinasios), All rights reserved.
Evolutionary Diversifaction of Aminopeptidase N in Lepidoptera by Conserved Clade-specific Amino Acid Residues

PubMed Central

Hughes, Austin L.

2015-01-01

Members of the aminopepidase N (APN) gene family of the insect order Lepidoptera (moths and butterflies) bind the naturally insecticidal Cry toxins produced by the bacterium Bacillus thuringiensis. Phylogenetic analysis of amino acid sequences of seven lepidopteran APN classes provided strong support for the hypothesis that lepidopteran APN2 class arose by gene duplication prior to the most recent common ancestor of Lepidoptera and Diptera. The Cry toxin-binding region (BR) of lepidopteran and dipteran APNs was subject to stronger purifying selection within APN classes than was the remainder of the molecule, reflecting conservation of catalytic site and adjoining residues within the BR. Of lepidopteran APN classes, APN2, APN6, and APN8 showed the strongest evidence of functional specialization, both in expression patterns and in the occurrence of conserved derived amino acid residues. The latter three APN classes also shared a convergently evolved conserved residue close to the catalytic site. APN8 showed a particularly strong tendency towards class-specific conserved residues, including one of the catalytic site residues in the BR and ten others in close vicinity to the catalytic site residues. The occurrence of class-specific sequences along with the conservation of enzymatic function is consistent with the hypothesis that the presence of Cry toxins in the environment has been a factor shaping the evolution of this multi-gene family. PMID:24675701
RELAX: detecting relaxed selection in a phylogenetic framework.

PubMed

Wertheim, Joel O; Murrell, Ben; Smith, Martin D; Kosakovsky Pond, Sergei L; Scheffler, Konrad

2015-03-01

Relaxation of selective strength, manifested as a reduction in the efficiency or intensity of natural selection, can drive evolutionary innovation and presage lineage extinction or loss of function. Mechanisms through which selection can be relaxed range from the removal of an existing selective constraint to a reduction in effective population size. Standard methods for estimating the strength and extent of purifying or positive selection from molecular sequence data are not suitable for detecting relaxed selection, because they lack power and can mistake an increase in the intensity of positive selection for relaxation of both purifying and positive selection. Here, we present a general hypothesis testing framework (RELAX) for detecting relaxed selection in a codon-based phylogenetic framework. Given two subsets of branches in a phylogeny, RELAX can determine whether selective strength was relaxed or intensified in one of these subsets relative to the other. We establish the validity of our test via simulations and show that it can distinguish between increased positive selection and a relaxation of selective strength. We also demonstrate the power of RELAX in a variety of biological scenarios where relaxation of selection has been hypothesized or demonstrated previously. We find that obligate and facultative γ-proteobacteria endosymbionts of insects are under relaxed selection compared with their free-living relatives and obligate endosymbionts are under relaxed selection compared with facultative endosymbionts. Selective strength is also relaxed in asexual Daphnia pulex lineages, compared with sexual lineages. Endogenous, nonfunctional, bornavirus-like elements are found to be under relaxed selection compared with exogenous Borna viruses. Finally, selection on the short-wavelength sensitive, SWS1, opsin genes in echolocating and nonecholocating bats is relaxed only in lineages in which this gene underwent pseudogenization; however, selection on the functional medium/long-wavelength sensitive opsin, M/LWS1, is found to be relaxed in all echolocating bats compared with nonecholocating bats. © The Author 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.
Identification of a potential tumor differentiation factor receptor candidate in prostate cancer cells.

PubMed

Sokolowska, Izabela; Woods, Alisa G; Gawinowicz, Mary Ann; Roy, Urmi; Darie, Costel C

2012-07-01

Tumor differentiation factor (TDF) is a pituitary protein that is secreted into the bloodstream and has an endocrine function. TDF and TDF-P1, a 20-residue peptide selected from the ORF of TDF, induce differentiation in human breast and prostate cancer cells, but not in other cells. TDF has no known mechanism of action. In our recent study, we identified heat shock 70 kDa proteins (HSP70s) as TDF receptors (TDF-Rs) in breast cancer cells. Therefore, we sought to investigate whether TDF-R candidates from prostate cancer cells are the same as those identified in breast cancer cells. Here, we used TDF-P1 to purify the potential TDF-R candidates by affinity purification chromatography from DU145 and PC3 steroid-resistant prostate cancer cells, LNCaP steroid-responsive prostate cancer cells, and nonprostate NG108 neuroblastoma and BLK CL.4 fibroblast-like cells. We identified the purified proteins by MS, and validated them by western blotting, immunofluorescence microscopy, immunoaffinity purification chromatography, and structural biology. We identified seven candidate proteins, of which three were from the HSP70 family. These three proteins were validated as potential TDF-R candidates in LNCaP steroid-responsive and in DU145 and PC3 steroid-resistant prostate cancer cells, but not in NG108 neuroblastoma and BLK CL.4 fibroblast-like cells. Our previous study and the current study suggest that GRP78, and perhaps HSP70s, are strong TDF-R candidates, and further suggest that TDF interacts with its receptors exclusively in breast and prostate cells, inducing cell differentiation through a novel, steroid-independent pathway. © 2012 The Authors Journal compilation © 2012 FEBS.
Comprehensive biometric, biochemical and histopathological assessment of nutrient deficiencies in gilthead sea bream fed semi-purified diets.

PubMed

Ballester-Lozano, Gabriel F; Benedito-Palos, Laura; Estensoro, Itziar; Sitjà-Bobadilla, Ariadna; Kaushik, Sadasivam; Pérez-Sánchez, Jaume

2015-09-14

Seven isoproteic and isolipidic semi-purified diets were formulated to assess specific nutrient deficiencies in sulphur amino acids (SAA), n-3 long-chain PUFA (n-3 LC-PUFA), phospholipids (PL), P, minerals (Min) and vitamins (Vit). The control diet (CTRL) contained these essential nutrients in adequate amounts. Each diet was allocated to triplicate groups of juvenile gilthead sea bream fed to satiety over an 11-week feeding trial period. Weight gain of n-3 LC-PUFA, P-Vit and PL-Min-SAA groups was 50, 60-75 and 80-85 % of the CTRL group, respectively. Fat retention was decreased by all nutrient deficiencies except by the Min diet. Strong effects on N retention were found in n-3 LC-PUFA and P fish. Combined anaemia and increased blood respiratory burst were observed in n-3 LC-PUFA fish. Hypoproteinaemia was found in SAA, n-3 LC-PUFA, PL and Vit fish. Derangements of lipid metabolism were also a common disorder, but the lipodystrophic phenotype of P fish was different from that of other groups. Changes in plasma levels of electrolytes (Ca, phosphate), metabolites (creatinine, choline) and enzyme activities (alkaline phosphatase) were related to specific nutrient deficiencies in PL, P, Min or Vit fish, whereas changes in circulating levels of growth hormone and insulin-like growth factor I primarily reflected the intensity of the nutritional stressor. Histopathological scoring of the liver and intestine segments showed specific nutrient-mediated changes in lipid cell vacuolisation, inflammation of intestinal submucosa, as well as the distribution and number of intestinal goblet and rodlet cells. These results contribute to define the normal range of variation for selected biometric, biochemical, haematological and histochemical markers.
The Involvement of Thaumatin-Like Proteins in Plant Food Cross-Reactivity: A Multicenter Study Using a Specific Protein Microarray

PubMed Central

Palacín, Arantxa; Rivas, Luis A.; Gómez-Casado, Cristina; Aguirre, Jacobo; Tordesillas, Leticia; Bartra, Joan; Blanco, Carlos; Carrillo, Teresa; Cuesta-Herranz, Javier; Bonny, José A. Cumplido; Flores, Enrique; García-Alvarez-Eire, Mar G.; García-Nuñez, Ignacio; Fernández, Francisco J.; Gamboa, Pedro; Muñoz, Rosa; Sánchez-Monge, Rosa; Torres, Maria; Losada, Susana Varela; Villalba, Mayte; Vega, Francisco; Parro, Victor; Blanca, Miguel; Salcedo, Gabriel; Díaz-Perales, Araceli

2012-01-01

Cross-reactivity of plant foods is an important phenomenon in allergy, with geographical variations with respect to the number and prevalence of the allergens involved in this process, whose complexity requires detailed studies. We have addressed the role of thaumatin-like proteins (TLPs) in cross-reactivity between fruit and pollen allergies. A representative panel of 16 purified TLPs was printed onto an allergen microarray. The proteins selected belonged to the sources most frequently associated with peach allergy in representative regions of Spain. Sera from two groups of well characterized patients, one with allergy to Rosaceae fruit (FAG) and another against pollens but tolerant to food-plant allergens (PAG), were obtained from seven geographical areas with different environmental pollen profiles. Cross-reactivity between members of this family was demonstrated by inhibition assays. Only 6 out of 16 purified TLPs showed noticeable allergenic activity in the studied populations. Pru p 2.0201, the peach TLP (41%), chestnut TLP (24%) and plane pollen TLP (22%) proved to be allergens of probable relevance to fruit allergy, being mainly associated with pollen sensitization, and strongly linked to specific geographical areas such as Barcelona, Bilbao, the Canary Islands and Madrid. The patients exhibited >50% positive response to Pru p 2.0201 and to chestnut TLP in these specific areas. Therefore, their recognition patterns were associated with the geographical area, suggesting a role for pollen in the sensitization of these allergens. Finally, the co-sensitizations of patients considering pairs of TLP allergens were analyzed by using the co-sensitization graph associated with an allergen microarray immunoassay. Our data indicate that TLPs are significant allergens in plant food allergy and should be considered when diagnosing and treating pollen-food allergy. PMID:22970164
The significant role of carboxylated carbonaceous fragments in the electrochemistry of carbon nanotubes.

PubMed

Ma, Xiao; Jia, Li; Zhang, Lu; Zhu, Liande

2014-04-01

Carbon nanotubes (CNTs) have been widely employed as electrode materials in diverse branches of electrochemistry, which are claimed to display dramatically improved electrochemical behaviour compared to the conventional carbon materials. But a series of recent publications have demonstrated that the electrocatalysis of CNTs might be due to the presence of some impurities, such as metallic catalysts, nanographitic particles and amorphous carbon. For this reason, CNTs are usually purified or treated with nitric acid or nitric and sulphuric acid prior to their versatile applications. However, the strong acidic and oxidative conditions are so aggressive that serious erosion of the tube structures has inevitably taken place, which creates defects on the sidewalls and gives rise to numerous molecular byproducts, commonly referred as carboxylated carbonaceous fragments (CCFs). The adsorption of CCFs on CNTs greatly alters the surface conditions of CNTs which may significantly impact on their electrochemical properties. To this end, we wish to disclose whether the electrocatalysis of the nitric acid purified CNTs is affected by the adsorption of the CCFs. Ascorbic acid (AA) and β-nicotinamide adenine dinucleotide (NADH) as selected as the targeting benchmarks that are known to be insensitive to the presence of metallic impurities, which may guarantee the preclusion of the promoting contributions from the metallic catalysts resident in CNTs. We have demonstrated that the electrocatalytic activities of the CNTs are actually dominated by the adsorbed CCFs generated during the acidic pre-treatment. After removal of the CCFs by base rinse, the electrocatalytic properties of CNTs are greatly deteriorated and degraded to the level similar to the conventional graphite powder. We believe this finding is particularly meaningful to uncover the mysterious electrocatalysis of CNTs. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Trans-species polymorphism at antimicrobial innate immunity cathelicidin genes of Atlantic cod and related species

PubMed Central

Árnason, Einar

2015-01-01

Natural selection, the most important force in evolution, comes in three forms. Negative purifying selection removes deleterious variation and maintains adaptations. Positive directional selection fixes beneficial variants, producing new adaptations. Balancing selection maintains variation in a population. Important mechanisms of balancing selection include heterozygote advantage, frequency-dependent advantage of rarity, and local and fluctuating episodic selection. A rare pathogen gains an advantage because host defenses are predominantly effective against prevalent types. Similarly, a rare immune variant gives its host an advantage because the prevalent pathogens cannot escape the host’s apostatic defense. Due to the stochastic nature of evolution, neutral variation may accumulate on genealogical branches, but trans-species polymorphisms are rare under neutrality and are strong evidence for balancing selection. Balanced polymorphism maintains diversity at the major histocompatibility complex (MHC) in vertebrates. The Atlantic cod is missing genes for both MHC-II and CD4, vital parts of the adaptive immune system. Nevertheless, cod are healthy in their ecological niche, maintaining large populations that support major commercial fisheries. Innate immunity is of interest from an evolutionary perspective, particularly in taxa lacking adaptive immunity. Here, we analyze extensive amino acid and nucleotide polymorphisms of the cathelicidin gene family in Atlantic cod and closely related taxa. There are three major clusters, Cath1, Cath2, and Cath3, that we consider to be paralogous genes. There is extensive nucleotide and amino acid allelic variation between and within clusters. The major feature of the results is that the variation clusters by alleles and not by species in phylogenetic trees and discriminant analysis of principal components. Variation within the three groups shows trans-species polymorphism that is older than speciation and that is suggestive of balancing selection maintaining the variation. Using Bayesian and likelihood methods positive and negative selection is evident at sites in the conserved part of the genes and, to a larger extent, in the active part which also shows episodic diversifying selection, further supporting the argument for balancing selection. PMID:26038731
Development of a water purifier for radioactive cesium removal from contaminated natural water by radiation-induced graft polymerization

NASA Astrophysics Data System (ADS)

Seko, Noriaki; Hoshina, Hiroyuki; Kasai, Noboru; Shibata, Takuya; Saiki, Seiichi; Ueki, Yuji

2018-02-01

Six years after the Fukushima-nuclear accident, the dissolved radioactive cesium (Cs) is now hardly detected in environmental natural waters. These natural waters are directly used as source of drinking and domestic waters in disaster-stricken areas in Fukushima. However, the possibility that some radioactive Cs adsorbed on soil or leaves will contaminate these natural waters during heavy rains or typhoon is always present. In order for the returning residents to live with peace of mind, it is important to demonstrate the safety of the domestic waters that they will use for their daily life. For this purpose, we have synthesized a material for selective removal of radioactive Cs by introducing ammonium 12-molybdophosphate (AMP) onto polyethylene nonwoven fabric through radiation-induced emulsion graft polymerization technique. Water purifiers filled with the grafted Cs adsorbent were installed in selected houses in Fukushima. The capability of the grafted adsorbent to remove Cs from domestic waters was evaluated for a whole year. The results showed that the tap water filtered through the developed water purifier contained no radioactive Cs, signifying the very effective adsorption performance of the developed grafted adsorbent. From several demonstrations, we have commercialized the water purifier named "KranCsair®". Furthermore, we have also developed a method for the mass production of the grafted nonwoven fabric. Using a 30 L grafting reactor, it was possible to produce the grafted nonwoven fabric with a suitable range of degree of grafting. When an irradiated roll of nonwoven trunk fabric with a length of 10 m and a width of 30 cm was set in the reactor filled with glycidyl methacrylate (GMA), AMP, Tween 80 monomer emulsion solution at 40 °C for 1 h, the difference of Dgs in the length and the width on roll of fabrics was negligible.
Selective recruitment of nuclear factors to productively replicating herpes simplex virus genomes.

PubMed

Dembowski, Jill A; DeLuca, Neal A

2015-05-01

Much of the HSV-1 life cycle is carried out in the cell nucleus, including the expression, replication, repair, and packaging of viral genomes. Viral proteins, as well as cellular factors, play essential roles in these processes. Isolation of proteins on nascent DNA (iPOND) was developed to label and purify cellular replication forks. We adapted aspects of this method to label viral genomes to both image, and purify replicating HSV-1 genomes for the identification of associated proteins. Many viral and cellular factors were enriched on viral genomes, including factors that mediate DNA replication, repair, chromatin remodeling, transcription, and RNA processing. As infection proceeded, packaging and structural components were enriched to a greater extent. Among the more abundant proteins that copurified with genomes were the viral transcription factor ICP4 and the replication protein ICP8. Furthermore, all seven viral replication proteins were enriched on viral genomes, along with cellular PCNA and topoisomerases, while other cellular replication proteins were not detected. The chromatin-remodeling complexes present on viral genomes included the INO80, SWI/SNF, NURD, and FACT complexes, which may prevent chromatinization of the genome. Consistent with this conclusion, histones were not readily recovered with purified viral genomes, and imaging studies revealed an underrepresentation of histones on viral genomes. RNA polymerase II, the mediator complex, TFIID, TFIIH, and several other transcriptional activators and repressors were also affinity purified with viral DNA. The presence of INO80, NURD, SWI/SNF, mediator, TFIID, and TFIIH components is consistent with previous studies in which these complexes copurified with ICP4. Therefore, ICP4 is likely involved in the recruitment of these key cellular chromatin remodeling and transcription factors to viral genomes. Taken together, iPOND is a valuable method for the study of viral genome dynamics during infection and provides a comprehensive view of how HSV-1 selectively utilizes cellular resources.
Anti-CDR3 Therapy for B-Cell Malignancies

DTIC Science & Technology

2013-10-01

1-5 in the report). The "Tomlinson" human antibody phage library will be used to pan for antibodies that bind these target CDR3s and not the parent...selection of phage to a test antigen. Successful expression will lead directly to the selection of CDR3-specific antibody-encoding phage : from which we... phage that bind to the purified candidate antibody and not to irrelevant antibodies. Phage are from single chain Fv library. Sequence phage that
Different evolutionary patterns of SNPs between domains and unassigned regions in human protein-coding sequences.

PubMed

Pang, Erli; Wu, Xiaomei; Lin, Kui

2016-06-01

Protein evolution plays an important role in the evolution of each genome. Because of their functional nature, in general, most of their parts or sites are differently constrained selectively, particularly by purifying selection. Most previous studies on protein evolution considered individual proteins in their entirety or compared protein-coding sequences with non-coding sequences. Less attention has been paid to the evolution of different parts within each protein of a given genome. To this end, based on PfamA annotation of all human proteins, each protein sequence can be split into two parts: domains or unassigned regions. Using this rationale, single nucleotide polymorphisms (SNPs) in protein-coding sequences from the 1000 Genomes Project were mapped according to two classifications: SNPs occurring within protein domains and those within unassigned regions. With these classifications, we found: the density of synonymous SNPs within domains is significantly greater than that of synonymous SNPs within unassigned regions; however, the density of non-synonymous SNPs shows the opposite pattern. We also found there are signatures of purifying selection on both the domain and unassigned regions. Furthermore, the selective strength on domains is significantly greater than that on unassigned regions. In addition, among all of the human protein sequences, there are 117 PfamA domains in which no SNPs are found. Our results highlight an important aspect of protein domains and may contribute to our understanding of protein evolution.
Purifying Selection on Exonic Splice Enhancers in Intronless Genes

PubMed Central

Savisaar, Rosina; Hurst, Laurence D.

2016-01-01

Exonic splice enhancers (ESEs) are short nucleotide motifs, enriched near exon ends, that enhance the recognition of the splice site and thus promote splicing. Are intronless genes under selection to avoid these motifs so as not to attract the splicing machinery to an mRNA that should not be spliced, thereby preventing the production of an aberrant transcript? Consistent with this possibility, we find that ESEs in putative recent retrocopies are at a higher density and evolving faster than those in other intronless genes, suggesting that they are being lost. Moreover, intronless genes are less dense in putative ESEs than intron-containing ones. However, this latter difference is likely due to the skewed base composition of intronless sequences, a skew that is in line with the general GC richness of few exon genes. Indeed, after controlling for such biases, we find that both intronless and intron-containing genes are denser in ESEs than expected by chance. Importantly, nucleotide-controlled analysis of evolutionary rates at synonymous sites in ESEs indicates that the ESEs in intronless genes are under purifying selection in both human and mouse. We conclude that on the loss of introns, some but not all, ESE motifs are lost, the remainder having functions beyond a role in splice promotion. These results have implications for the design of intronless transgenes and for understanding the causes of selection on synonymous sites. PMID:26802218
40 CFR 1065.307 - Linearity verification.

Code of Federal Regulations, 2010 CFR

2010-07-01

... different flow rates. Use a gravimetric reference measurement (such as a scale, balance, or mass comparator... the gas-division system to divide the span gas with purified air or nitrogen. Select gas divisions... PM balance, m max refers to the typical mass of a PM filter. (ii) For linearity verification of...
Host-parasite interactions and purifying selection in a microsporidian parasite of honey bees

USDA-ARS?s Scientific Manuscript database

Nosema ceranae is a microsporidian parasite that infects honey bee mid-gut epithelial cells. Infection can impair honey bee cognitive function, shorten lifespan, suppress the innate immune response and inhibit the apoptosis of infected gut cells. However, the virulence factors of this parasite are s...
Method for removal of phosgene from boron trichloride

DOEpatents

Freund, Samuel M.

1983-01-01

Selective ultraviolet photolysis using an unfiltered mercury arc lamp has been used to substantially reduce the phosgene impurity in a mixture of boron trichloride and phosgene. Infrared spectrophotometric analysis of the sample before and after irradiation shows that is is possible to highly purify commercially available boron trichloride with this method.

PURIFICATION AND PARTIAL CHARACTERIZATION OF AN ACID PHOSPHATASE FROM SPIRODELA OLIGORRHIZA AND ITS AFFINITY FOR SELECTED ORGANOPHOSPHATE PESTICIDES

EPA Science Inventory

An acid phosphatase from the aquatic plant Spirodela oligorrhiza (duckweed) was isolated by fast protein liquid chromatography (FPLC) and partially characterized. The enzyme was purified 1871-fold with a total yield of 40%. SDS-PAGE electrophoresis of the pure acid phosphatase ...
Determining the Effect of Natural Selection on Linked Neutral Divergence across Species

PubMed Central

Phung, Tanya N.; Lohmueller, Kirk E.

2016-01-01

A major goal in evolutionary biology is to understand how natural selection has shaped patterns of genetic variation across genomes. Studies in a variety of species have shown that neutral genetic diversity (intra-species differences) has been reduced at sites linked to those under direct selection. However, the effect of linked selection on neutral sequence divergence (inter-species differences) remains ambiguous. While empirical studies have reported correlations between divergence and recombination, which is interpreted as evidence for natural selection reducing linked neutral divergence, theory argues otherwise, especially for species that have diverged long ago. Here we address these outstanding issues by examining whether natural selection can affect divergence between both closely and distantly related species. We show that neutral divergence between closely related species (e.g. human-primate) is negatively correlated with functional content and positively correlated with human recombination rate. We also find that neutral divergence between distantly related species (e.g. human-rodent) is negatively correlated with functional content and positively correlated with estimates of background selection from primates. These patterns persist after accounting for the confounding factors of hypermutable CpG sites, GC content, and biased gene conversion. Coalescent models indicate that even when the contribution of ancestral polymorphism to divergence is small, background selection in the ancestral population can still explain a large proportion of the variance in divergence across the genome, generating the observed correlations. Our findings reveal that, contrary to previous intuition, natural selection can indirectly affect linked neutral divergence between both closely and distantly related species. Though we cannot formally exclude the possibility that the direct effects of purifying selection drive some of these patterns, such a scenario would be possible only if more of the genome is under purifying selection than currently believed. Our work has implications for understanding the evolution of genomes and interpreting patterns of genetic variation. PMID:27508305
Determining the Effect of Natural Selection on Linked Neutral Divergence across Species.

PubMed

Phung, Tanya N; Huber, Christian D; Lohmueller, Kirk E

2016-08-01

A major goal in evolutionary biology is to understand how natural selection has shaped patterns of genetic variation across genomes. Studies in a variety of species have shown that neutral genetic diversity (intra-species differences) has been reduced at sites linked to those under direct selection. However, the effect of linked selection on neutral sequence divergence (inter-species differences) remains ambiguous. While empirical studies have reported correlations between divergence and recombination, which is interpreted as evidence for natural selection reducing linked neutral divergence, theory argues otherwise, especially for species that have diverged long ago. Here we address these outstanding issues by examining whether natural selection can affect divergence between both closely and distantly related species. We show that neutral divergence between closely related species (e.g. human-primate) is negatively correlated with functional content and positively correlated with human recombination rate. We also find that neutral divergence between distantly related species (e.g. human-rodent) is negatively correlated with functional content and positively correlated with estimates of background selection from primates. These patterns persist after accounting for the confounding factors of hypermutable CpG sites, GC content, and biased gene conversion. Coalescent models indicate that even when the contribution of ancestral polymorphism to divergence is small, background selection in the ancestral population can still explain a large proportion of the variance in divergence across the genome, generating the observed correlations. Our findings reveal that, contrary to previous intuition, natural selection can indirectly affect linked neutral divergence between both closely and distantly related species. Though we cannot formally exclude the possibility that the direct effects of purifying selection drive some of these patterns, such a scenario would be possible only if more of the genome is under purifying selection than currently believed. Our work has implications for understanding the evolution of genomes and interpreting patterns of genetic variation.
Molecular evolution of the betagamma lens crystallin superfamily: evidence for a retained ancestral function in gamma N crystallins?

PubMed

Weadick, Cameron J; Chang, Belinda S W

2009-05-01

Within the vertebrate eye, betagamma crystallins are extremely stable lens proteins that are uniquely adapted to increase refractory power while maintaining transparency. Unlike alpha crystallins, which are well-characterized, multifunctional proteins that have important functions both in and out of the lens, betagamma lens crystallins are a diverse group of proteins with no clear ancestral or contemporary nonlens role. We carried out phylogenetic and molecular evolutionary analyses of the betagamma-crystallin superfamily in order to study the evolutionary history of the gamma N crystallins, a recently discovered, biochemically atypical family suggested to possess a divergent or ancestral function. By including nonlens, betagamma-motif-containing sequences in our analysis as outgroups, we confirmed the phylogenetic position of the gamma N family as sister to other gamma crystallins. Using maximum likelihood codon models to estimate lineage-specific nonsynonymous-to-synonymous rate ratios revealed strong positive selection in all of the early lineages within the betagamma family, with the striking exception of the lineage leading to the gamma N crystallins which was characterized by strong purifying selection. Branch-site analysis, used to identify candidate sites involved in functional divergence between gamma N crystallins and its sister clade containing all other gamma crystallins, identified several positively selected changes at sites of known functional importance in the betagamma crystallin protein structure. Further analyses of a fish-specific gamma N crystallin gene duplication revealed a more recent episode of positive selection in only one of the two descendant lineages (gamma N2). Finally, from the guppy, Poecilia reticulata, we isolated complete gamma N1 and gamma N2 coding sequence data from cDNA and partial coding sequence data from genomic DNA in order to confirm the presence of a novel gamma N2 intron, discovered through data mining of two pufferfish genomes. We conclude that the function of the gamma N family likely resembles the ancestral vertebrate betagamma crystallin more than other betagamma families. Furthermore, owing to the presence of an additional intron in some fish gamma N2 crystallins, and the inferred action of positive selection following the fish-specific gamma N duplication, we suggest that further study of fish gamma N crystallins will be critical in further elucidating possible ancestral functions of gamma N crystallins and any nonstructural role they may have.
The Rise and Fall of an Evolutionary Innovation: Contrasting Strategies of Venom Evolution in Ancient and Young Animals

PubMed Central

Sunagar, Kartik; Moran, Yehu

2015-01-01

Animal venoms are theorized to evolve under the significant influence of positive Darwinian selection in a chemical arms race scenario, where the evolution of venom resistance in prey and the invention of potent venom in the secreting animal exert reciprocal selection pressures. Venom research to date has mainly focused on evolutionarily younger lineages, such as snakes and cone snails, while mostly neglecting ancient clades (e.g., cnidarians, coleoids, spiders and centipedes). By examining genome, venom-gland transcriptome and sequences from the public repositories, we report the molecular evolutionary regimes of several centipede and spider toxin families, which surprisingly accumulated low-levels of sequence variations, despite their long evolutionary histories. Molecular evolutionary assessment of over 3500 nucleotide sequences from 85 toxin families spanning the breadth of the animal kingdom has unraveled a contrasting evolutionary strategy employed by ancient and evolutionarily young clades. We show that the venoms of ancient lineages remarkably evolve under the heavy constraints of negative selection, while toxin families in lineages that originated relatively recently rapidly diversify under the influence of positive selection. We propose that animal venoms mostly employ a ‘two-speed’ mode of evolution, where the major influence of diversifying selection accompanies the earlier stages of ecological specialization (e.g., diet and range expansion) in the evolutionary history of the species–the period of expansion, resulting in the rapid diversification of the venom arsenal, followed by longer periods of purifying selection that preserve the potent toxin pharmacopeia–the period of purification and fixation. However, species in the period of purification may re-enter the period of expansion upon experiencing a major shift in ecology or environment. Thus, we highlight for the first time the significant roles of purifying and episodic selections in shaping animal venoms. PMID:26492532
Synapsin I is associated with cholinergic nerve terminals in the electric organs of Torpedo, Electrophorus, and Malapterurus and copurifies with Torpedo synaptic vesicles.

PubMed

Volknandt, W; Naito, S; Ueda, T; Zimmermann, H

1987-08-01

Using an affinity-purified monospecific polyclonal antibody against bovine brain synapsin I, the distribution of antigenically related proteins was investigated in the electric organs of the three strongly electric fish Torpedo marmorata, Electrophorus electricus, Malapterurus electricus and in the rat diaphragm. On application of indirect fluorescein isothiocyanate-immunofluorescence and using alpha-bungarotoxin for identification of synaptic sites, intense and very selective staining of nerve terminals was found in all of these tissues. Immunotransfer blots of tissue homogenates revealed specific bands whose molecular weights are similar to those of synapsin Ia and synapsin Ib. Moreover, synapsin I-like proteins are still attached to the synaptic vesicles that were isolated in isotonic glycine solution from Torpedo electric organ by density gradient centrifugation and chromatography on Sephacryl-1000. Our results suggest that synapsin I-like proteins are also associated with cholinergic synaptic vesicles of electric organs and that the electric organ may be an ideal source for studying further the functional and molecular properties of synapsin.
Functional display of family 11 endoxylanases on the surface of phage M13.

PubMed

Beliën, T; Hertveldt, K; Van den Brande, K; Robben, J; Van Campenhout, S; Volckaert, G

2005-02-09

Two family 11 endoxylanases (EC 3.2.1.8) were functionally displayed on the surface of bacteriophage M13. The genes encoding endo-1,4-xylanase I from Aspergillus niger (ExlA) and endo-1,4-xylanase A from Bacillus subtilis (XynA) were fused to the gene encoding the minor coat protein g3p in phagemid vector pHOS31. Phage rescue resulted in functional monovalent display of the enzymes as was demonstrated by three independent tests. Firstly, purified recombinant phage particles showed a clear hydrolytic activity in an activity assay based on insoluble, chromagenic arabinoxylan substrate. Secondly, specific binding of endoxylanase displaying phages to immobilized endoxylanase inhibitors was demonstrated by interaction ELISA. Finally, two rounds of selection and amplification in a biopanning procedure against immobilized endoxylanase inhibitor were performed. Phages displaying endoxylanases were strongly enriched from background phages displaying unrelated proteins. These results open perspectives to use phage display for analysing protein-protein interactions at the interface between endoxylanases and their inhibitors. In addition, this technology should enable engineering of endoxylanases into novel variants with altered binding properties towards endoxylanase inhibitors.
Sustainable efficient way for opioid peptide LVV-h7 preparation from enzymatic proteolysis in a microfluidic-based reaction-extraction process with solvent recycling.

PubMed

Elagli, Adil; Belhacene, Kalim; Dhulster, Pascal; Froidevaux, Renato

2016-05-01

LVV-h7 (LVVYPWTQFR) is a bioactive peptide that can be obtained from blood as waste of food industry, more precisely from hemoglobin hydrolysis by pepsin. This opioid peptide belongs to the hemorphins family and have strong physiological effects that bring its use in pharmaceutics and various therapeutic treatments attractive, in particular for substituting its costly chemically synthetized analogous. Hemoglobin hydrolysis by pepsin generates a huge variety of peptides among whose LVV-h7 can be purified by liquid-liquid extraction (LLE). Herein, selective preparation of this peptide is proposed by a microfluidic-based continuous reaction-separation process. Hemoglobin hydrolysis in microreactor was firstly coupled to LVV-h7 LLE in octan-1-ol and then coupled to LVV-h7 back LLE in acidic water. This continuous process allowed to prepare pure LVV-h7, as confirmed by liquid chromatography and mass spectrometry. The microfluidic circuit also allowed octan-1-ol recycling in a closed loop, making this method more sustainable than similar biphasic batch process. Copyright © 2016 Elsevier B.V. All rights reserved.
A specific DNA probe which identifies Babesia bovis in whole blood.

PubMed

Petchpoo, W; Tan-ariya, P; Boonsaeng, V; Brockelman, C R; Wilairat, P; Panyim, S

1992-05-01

A genomic library of Babesia bovis DNA from the Mexican strain M was constructed in plasmid pUN121 and cloned in Escherichia coli. Several recombinants which hybridized strongly to radioactively labeled B. bovis genomic DNA in an in situ screening were selected and further analyzed for those which specifically hybridized to B. bovis DNA. It was found that pMU-B1 had the highest sensitivity, detecting 25 pg of purified B. bovis DNA, and 300 parasites in 10 microliters of whole infected blood, or 0.00025% parasitemia. pMU-B1 contained a 6.0 kb B. bovis DNA insert which did not cross-hybridize to Babesia bigemina, Trypanosoma evansi, Plasmodium falciparum, Anaplasma marginale, Boophilus microplus and cow DNA. In the Southern blot analysis of genomic DNA, pMU-B1 could differentiate between two B. bovis geographic isolates, Mexican strain M and Thai isolate TS4. Thus, the pMU-B1 probe will be useful in the diagnosis of Babesia infection in cattle and ticks, and in the differentiation of B. bovis strains.
Catalyst-Free Growth of Three-Dimensional Graphene Flakes and Graphene/g-C₃N₄ Composite for Hydrocarbon Oxidation.

PubMed

Chen, Ke; Chai, Zhigang; Li, Cong; Shi, Liurong; Liu, Mengxi; Xie, Qin; Zhang, Yanfeng; Xu, Dongsheng; Manivannan, Ayyakkannu; Liu, Zhongfan

2016-03-22

Mass production of high-quality graphene flakes is important for commercial applications. Graphene microsheets have been produced on an industrial scale by chemical and liquid-phase exfoliation of graphite. However, strong-interaction-induced interlayer aggregation usually leads to the degradation of their intrinsic properties. Moreover, the crystallinity or layer-thickness controllability is not so perfect to fulfill the requirement for advanced technologies. Herein, we report a quartz-powder-derived chemical vapor deposition growth of three-dimensional (3D) high-quality graphene flakes and demonstrate the fabrication and application of graphene/g-C3N4 composites. The graphene flakes obtained after the removal of growth substrates exhibit the 3D curved microstructure, controllable layer thickness, good crystallinity, as well as weak interlayer interactions suitable for preventing the interlayer stacking. Benefiting from this, we achieved the direct synthesis of g-C3N4 on purified graphene flakes to form the uniform graphene/g-C3N4 composite, which provides efficient electron transfer interfaces to boost its catalytic oxidation activity of cycloalkane with relatively high yield, good selectivity, and reliable stability.
Rapid molecular evolution of human bocavirus revealed by Bayesian coalescent inference.

PubMed

Zehender, Gianguglielmo; De Maddalena, Chiara; Canuti, Marta; Zappa, Alessandra; Amendola, Antonella; Lai, Alessia; Galli, Massimo; Tanzi, Elisabetta

2010-03-01

Human bocavirus (HBoV) is a linear single-stranded DNA virus belonging to the Parvoviridae family that has recently been isolated from the upper respiratory tract of children with acute respiratory infection. All of the strains observed so far segregate into two genotypes (1 and 2) with a low level of polymorphism. Given the recent description of the infection and the lack of epidemiological and molecular data, we estimated the virus's rates of molecular evolution and population dynamics. A dataset of forty-nine dated VP2 sequences, including also eight new isolates obtained from pharyngeal swabs of Italian patients with acute respiratory tract infections, was submitted to phylogenetic analysis. The model parameters, evolutionary rates and population dynamics were co-estimated using a Bayesian Markov Chain Monte Carlo approach, and site-specific positive and negative selection was also investigated. Recombination was investigated by seven different methods and one suspected recombinant strain was excluded from further analysis. The estimated mean evolutionary rate of HBoV was 8.6x10(-4)subs/site/year, and that of the 1st+2nd codon positions was more than 15 times less than that of the 3rd codon position. Viral population dynamics analysis revealed that the two known genotypes diverged recently (mean tMRCA: 24 years), and that the epidemic due to HBoV genotype 2 grew exponentially at a rate of 1.01year(-1). Selection analysis of the partial VP2 showed that 8.5% of sites were under significant negative pressure and the absence of positive selection. Our results show that, like other parvoviruses, HBoV is characterised by a rapid evolution. The low level of polymorphism is probably due to a relatively recent divergence between the circulating genotypes and strong purifying selection acting on viral antigens.
Molecular evolutionary analysis of vertebrate transducins: a role for amino acid variation in photoreceptor deactivation.

PubMed

Lin, Yi G; Weadick, Cameron J; Santini, Francesco; Chang, Belinda S W

2013-12-01

Transducin is a heterotrimeric G protein that plays a critical role in phototransduction in the rod and cone photoreceptor cells of the vertebrate retina. Rods, highly sensitive cells that recover from photoactivation slowly, underlie dim-light vision, whereas cones are less sensitive, recover more quickly, and underlie bright-light vision. Transducin deactivation is a critical step in photoreceptor recovery and may underlie the functional distinction between rods and cones. Rods and cones possess distinct transducin α subunits, yet they share a common deactivation mechanism, the GTPase activating protein (GAP) complex. Here, we used codon models to examine patterns of sequence evolution in rod (GNAT1) and cone (GNAT2) α subunits. Our results indicate that purifying selection is the dominant force shaping GNAT1 and GNAT2 evolution, but that GNAT2 has additionally been subject to positive selection operating at multiple phylogenetic scales; phylogeny-wide analysis identified several sites in the GNAT2 helical domain as having substantially elevated dN/dS estimates, and branch-site analysis identified several nearby sites as targets of strong positive selection during early vertebrate history. Examination of aligned GNAT and GAP complex crystal structures revealed steric clashes between several positively selected sites and the deactivating GAP complex. This suggests that GNAT2 sequence variation could play an important role in adaptive evolution of the vertebrate visual system via effects on photoreceptor deactivation kinetics and provides an alternative perspective to previous work that focused instead on the effect of GAP complex concentration. Our findings thus further the understanding of the molecular biology, physiology, and evolution of vertebrate visual systems.
A Novel Point Mutation at Position 156 of Reverse Transcriptase from Feline Immunodeficiency Virus Confers Resistance to the Combination of (−)-β-2′,3′-Dideoxy-3′-Thiacytidine and 3′-Azido-3′-Deoxythymidine

PubMed Central

Smith, Robert A.; Remington, Kathryn M.; Preston, Bradley D.; Schinazi, Raymond F.; North, Thomas W.

1998-01-01

Mutants of feline immunodeficiency virus (FIV) resistant to (−)-β-2′,3′-dideoxy-3′-thiacytidine (3TC) were selected by culturing virus in the presence of increasing stepwise concentrations of 3TC. Two plaque-purified variants were isolated from the original mutant population, and both of these mutants were resistant to 3TC. Surprisingly, these mutants were also phenotypically resistant to 3′-azido-3′-deoxythymidine (AZT) and to the combination of 3TC and AZT. Purified reverse transcriptase (RT) from one of these plaque-purified mutants was resistant to the 5′-triphosphates of 3TC and AZT. DNA sequence analysis of the RT-encoding region of the pol gene amplified from the plaque-purified mutants revealed a Pro-to-Ser mutation at position 156 of RT. A site-directed mutant of FIV engineered to contain this Pro-156-Ser mutation was resistant to 3TC, AZT, and the combination of 3TC and AZT, confirming the role of the Pro-156-Ser mutation in the resistance of FIV to these two nucleoside analogs. This represents the first report of a lentiviral mutant resistant to the combination of AZT and 3TC due to a single, unique point mutation. PMID:9499094
Isolation of friedelin from black condensate of cork.

PubMed

Pires, Ricardo A; Aroso, Ivo; Silva, Susana P; Mano, João F; Reis, Rui L

2011-11-01

Black condensates (BC) are wastes of the insulation corkboard industry that contain several valuable chemicals, including friedelin, a terpene exhibiting biological activity. Herein, we report a straightforward procedure to extract friedelin from BC. Using this procedure, we were able to extract friedelin with yields between 0.4% and 2.9% and to further purify it obtaining purities from 77.0% to 99.3% (HPLC). The initial BC (2 batches), extracted raw product and purified friedelin were analyzed using FTIR. The extraction yields and purities were found to be directly related to the intensity of the carbonyl vibration at 1713 cm(-1) in the FTIR spectrum of the used BC batch. Therefore, these spectra can be used to screen and select BC batches suitable for friedelin extraction.
Expression, purification, and characterization of an enzymatically active truncated human rho-kinase I (ROCK I) domain expressed in Sf-9 insect cells.

PubMed

Khandekar, Sanjay S; Yi, Tracey; Dul, Ed; Wright, Lois L; Chen, Susan; Scott, Gilbert F; Smith, Gary K; Lee, Dennis; Hu, Erding; Kirkpatrick, Robert B

2006-01-01

Rho Kinase I (ROCK I) is a serine/threonine kinase that is involved in diverse cellular signaling. To further understand the physiological role of ROCK I and to identify and develop potent and selective inhibitors of ROCK I, we have overexpressed and purified a constitutively active dimeric human ROCK I (3-543) kinase domain using the Sf9-baculovirus expression system. In addition, using a limited proteolysis technique, we have identified a minimal functional subdomain of ROCK I that can be used in crystallization studies. The availability of multimilligram amounts of purified and well characterized functional human ROCK I kinase domains will be useful in screening and structural studies.
Lectins discriminate between pathogenic and nonpathogenic South American trypanosomes

DOE Office of Scientific and Technical Information (OSTI.GOV)

de Miranda Santos, I.K.; Pereira, M.E.

1984-09-01

Cell surface carbohydrates of Trypanosoma cruzi, Trypanosoma rangeli, and Trypanosoma conorhini were analyzed by a micro-agglutination assay employing 27 highly purified lectins and by binding assays using various /sup 125/I-labeled lectins. The following seven lectins discriminated between the trypanosomes: 1) tomato lectin (an N-acetyl-D-glucosamine-binding protein), both in purified form and as crude tomato juice; 2) Bauhinea purpurea and Sophora japonica lectins (both N-acetyl-D-galactosamine-binding proteins), which selectively agglutinated T. cruzi; 3) Vicia villosa (an N-acetyl-D-galactosamine-binding protein) which was specific for T. rangeli; 4) peanut lectin (a D-galactose-binding protein) both in purified form and as crude saline extract; and 5) Ulex europaeusmore » and Lotus tetragonolobus (both L-fucose-binding proteins) lectins which reacted only with T. conorhini. Binding studies with 125I-labeled lectins were performed to find whether unagglutinated cells of the three different species of trypanosomes might have receptors for these lectins, in which case absence of agglutination could be due to a peculiar arrangement of the receptors. These assays essentially confirmed the agglutination experiments.« less
Brevibacillus laterosporus strain BPM3, a potential biocontrol agent isolated from a natural hot water spring of Assam, India.

PubMed

Saikia, R; Gogoi, D K; Mazumder, S; Yadav, A; Sarma, R K; Bora, T C; Gogoi, B K

2011-03-20

A bacterial strain designated as BPM3 isolated from mud of a natural hot water spring of Nambar Wild Life Sanctuary, Assam, India, strongly inhibited growth of phytopathogenic fungi (Fusarium oxysporum f. sp. ciceri, F. semitectum, Magnaporthe grisea and Rhizoctonia oryzae) and gram-positive bacterium (Staphylococcus aureus). The maximum growth and antagonistic activity was recorded at 30°C, pH 8.5 when starch and peptone were amended as carbon and nitrogen sources, respectively. In greenhouse experiment, this bacterium (BPM3) suppressed blast disease of rice by 30-67% and protected the weight loss by 35-56.5%. The maximum disease protection (67%) and weight loss protection (56.5%) were recorded when the bacterium was applied before 2 days of the pathogen inoculation. Antifungal and antibacterial compounds were isolated from the bacterium which also inhibited the growth of these targeted pathogens. The compounds were purified and on spectroscopic analysis of a purified fraction having R(f) 0.22 which showed strong antifungal and antibacterial activity indicated the presence of C-H, carbonyl group, dimethyl group, -CH(2) and methyl group. The bacterium was characterized by morphological, biochemical and molecular approaches and confirmed that the strain BPM3 is Brevibacillus laterosporus. Copyright © 2010 Elsevier GmbH. All rights reserved.
Anti-oxidative and Anti-microbial Activities of Purified MPN-1-1 from Persicaria nepalensis (Meisn.) Miyabe.

PubMed

Yang, Woong-Suk; Yang, Seung-Hoon; Lee, Jae-Yong; Jang, Seong-Ho; Kim, Cheorl-Ho; Hwnag, Cher-Won

2017-01-01

Persicaria is a genus of flowering plants generally used for traditional medicine and nutritional supplements in tropical and subtropical East Asian countries. Previous studies have shown that Persicaria extracts alleviate lipid peroxidation, hypertension, and inflammation. We investigated the anti-oxidative and anti-microbial effects of ethanol extracts of Persicaria nepalensis (Meisn.) Miyabe, and isolated and identified an active compound, MPN-1-1 from the ethanol extracts. Anti-oxidative values, as indicated by the Oxygen Radical Absorbance Capacity (ORAC) assay, were enhanced by treatment with Persicaria nepalensis (Meisn.) Miyabe ethanol extracts, and bacterial growth was inhibited. The active compound (MPN-1-1), which was further isolated and purified from a Persicaria nepalensis (Meisn.) Miyabe ethanol extract by medium pressure liquid chromatography (MPLC), also had strong anti-oxidative and anti-microbial activity. 1H-NMR spectroscopy identified MPN-1-1 as a 1-ethenyl-4,8-dimethoxy-9H-pyrido(3,4-β) indole compound, which is an alkaloid. Our results provide evidence that Persicaria nepalensis (Meisn.) Miyabe extract has strong physiological activity without any toxic effects, and furthermore, MPN-1-1 can be potentially utilized as a natural dietary supplement as well as an anti-oxidant. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.
TLR8-driven IL-12-dependent reciprocal and synergistic activation of NK cells and monocytes by immunostimulatory RNA.

PubMed

Berger, Michael; Ablasser, Andrea; Kim, Sarah; Bekeredjian-Ding, Isabelle; Giese, Thomas; Endres, Stefan; Hornung, Veit; Hartmann, Gunther

2009-04-01

Immunostimulatory RNA (isRNA) depending on sequence and structure can function as a ligand for Toll-like receptor (TLR) 7 and TLR8. Here we show that isRNA induces high levels of bioactive interleukin-12 in purified human monocytes, whereas purified natural killer (NK) cells did not respond. However, in a coculture of monocytes and NK cells, isRNA dramatically increased NK cell function. Activation of monocytes and NK cells was bidirectional, as monocytes in the presence of NK cells produced higher levels of bioactive interleukin-12. As a result of the monocyte-NK cell interaction in peripheral blood mononuclear cells isRNA induced high levels of interferon (IFN)-gamma in NK cells and strong NK cell-mediated cytotoxic activity. Induction of simultaneous IFN-gamma production and lytic activity by isRNA in NK cells was higher as compared with other established nucleic acid or small molecule TLR ligands. Our studies demonstrate that monocytes play a pivotal role in the orchestration of a strong NK cell response. With early NK cell-dependent IFN-gamma production being critical for the development of antigen-specific cytotoxic T lymphocyte responses, newly developed isRNA-based TLR8 ligands join the list of promising oligonucleotides for immunotherapy of viral infection and cancer.
Purification and characterization of a thermostable α-galactosidase from Thielavia terrestris NRRL 8126 in solid state fermentation.

PubMed

Saad, Rawia R; Fawzi, Eman M

2012-03-01

Several seeds and husks of some plants belonging to leguminosae, Graminae, Compositae and Palmae were evaluated as carbon substrates to produce α-galactosidase (α-Gal) by the thermophilic fungus, Thielavia terrestris NRRL 8126 in solid substrate fermentation. The results showed that Cicer arietinum (chick pea seed) was the best substrate for α-Gal production. The crude enzyme was precipitated by ammonium sulphate (60%) and purified by gel filtration on sephadex G-100 followed by ion exchange chromatography on DEAE-Cellulose. The final purification fold of the enzyme was 30.42. The temperature and pH optima of purified α-Gal from Thielavia terrestris were 70 °C and 6.5, respectively. The enzyme showed high thermal stability at 70 °C and 75 °C and the half-life of the α-Gal at 90 °C was 45 min. Km of the purified enzyme was 1.31 mM. The purified enzyme was inhibited by Ag2+, Hg2+, Zn2+, Ba2+, Mg2+, Mn2+ and Fe2+ at 5 mM and 10 mM. Also, EDTA, sodium arsenate, L-cysteine and iodoacetate inhibited the enzyme activity. On the other hand, Ca2+, Cu2+, K+ and Na+ slightly enhanced the enzyme activity at 5 mM while at 10 mM they caused inhibition. The molecular weight of the α-Gal was estimated to be 82 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This enzyme displays a number of biochemical properties that make it a potentially strong candidate for biotechnological and medicinal applications.

Identification and immunologic characterization of an allergen, alliin lyase, from garlic (Allium sativum).

PubMed

Kao, Shao-Hsuan; Hsu, Ching-Hsian; Su, Song-Nan; Hor, Wei-Ting; Chang T, Wen-Hong; Chow, Lu-Ping

2004-01-01

Garlic (Allium sativum) is one of the most common relishes used in cooking worldwide. Very few garlic allergens have been reported, and garlic allergy has been rarely studied. The aim of the study was to identify allergenic proteins in garlic and to investigate their importance in allergies to other Allium species (leek, shallot, and onion). A crude extract of garlic proteins was separated by SDS-PAGE and 2-dimensional electrophoresis; immunoblotting was then performed with the use of individual and pooled sera from patients with garlic allergy, and the major IgE-binding proteins were analyzed by amino acid sequencing and mass spectrometry. The putative allergens were further purified by chromatography; the antigenicity, allergenicity, and IgE-binding cross-reactivity of the purified protein were then studied by immunoblotting, periodate oxidation, skin tests, and IgE-binding inhibition assays. A major allergen, alliin lyase, was identified by mass spectrometry and Edman sequencing and purified to homogeneity through the use of a simple 2-step chromatographic method. Skin tests showed that the purified protein elicited IgE-mediated hypersensitive responses in patients with garlic allergy. Periodate oxidation showed that carbohydrate groups were involved in the antigenicity, allergenicity, and cross-reactivity. Garlic alliin lyase showed strong cross-reactivity with alliin lyases from other Allium species, namely leek, shallot, and onion. Alliin lyase was found to be a major garlic allergen in a garlic-allergic group of patients in Taiwan. The wide distribution of alliin lyase in Allium suggests it may be a new cross-reactive allergen.
Purification and characterization of a novel neurotensin-degrading peptidase from rat brain synaptic membranes.

PubMed

Checler, F; Vincent, J P; Kitabgi, P

1986-08-25

A peptidase that cleaved neurotensin at the Pro10-Tyr11 peptide bond, leading to the formation of neurotensin-(1-10) and neurotensin-(11-13), was purified nearly to homogeneity from rat brain synaptic membranes. The enzyme appeared to be monomeric with a molecular weight of about 70,000-75,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and high pressure liquid chromatography filtration. Isoelectrofocusing indicated a pI of 5.9-6. The purified peptidase could be classified as a neutral metallopeptidase with respect to its sensitivity to pH and metal chelators. Thiol-blocking agents and acidic and serine protease inhibitors had no effect. Studies with specific peptidase inhibitors clearly indicated that the purified enzyme was distinct from enzymes capable of cleaving neurotensin at the Pro10-Tyr11 bond such as proline endopeptidase and endopeptidase 24-11. The enzyme was also distinct from other neurotensin-degrading peptidases such as angiotensin-converting enzyme and a recently purified rat brain soluble metalloendopeptidase. The peptidase displayed a high affinity for neurotensin (Km = 2.6 microM). Studies on its specificity revealed that neurotensin-(9-13) was the shortest neurotensin partial sequence that was able to fully inhibit [3H]neurotensin degradation. Shortening the C-terminal end of the neurotensin molecule as well as substitutions in positions 8, 9, and 11 by D-amino acids strongly decreased the inhibitory potency of neurotensin. Among 20 natural peptides, only angiotensin I and the neurotensin-related peptides (xenopsin and neuromedin N) were found as potent as unlabeled neurotensin.
Isolation and characterization of an RIP (ribosome-inactivating protein)-like protein from tobacco with dual enzymatic activity.

PubMed

Sharma, Neelam; Park, Sang-Wook; Vepachedu, Ramarao; Barbieri, Luigi; Ciani, Marialibera; Stirpe, Fiorenzo; Savary, Brett J; Vivanco, Jorge M

2004-01-01

Ribosome-inactivating proteins (RIPs) are N-glycosidases that remove a specific adenine from the sarcin/ricin loop of the large rRNA, thus arresting protein synthesis at the translocation step. In the present study, a protein termed tobacco RIP (TRIP) was isolated from tobacco (Nicotiana tabacum) leaves and purified using ion exchange and gel filtration chromatography in combination with yeast ribosome depurination assays. TRIP has a molecular mass of 26 kD as evidenced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and showed strong N-glycosidase activity as manifested by the depurination of yeast rRNA. Purified TRIP showed immunoreactivity with antibodies of RIPs from Mirabilis expansa. TRIP released fewer amounts of adenine residues from ribosomal (Artemia sp. and rat ribosomes) and non-ribosomal substrates (herring sperm DNA, rRNA, and tRNA) compared with other RIPs. TRIP inhibited translation in wheat (Triticum aestivum) germ more efficiently than in rabbit reticulocytes, showing an IC50 at 30 ng in the former system. Antimicrobial assays using highly purified TRIP (50 microg mL(-1)) conducted against various fungi and bacterial pathogens showed the strongest inhibitory activity against Trichoderma reesei and Pseudomonas solancearum. A 15-amino acid internal polypeptide sequence of TRIP was identical with the internal sequences of the iron-superoxide dismutase (Fe-SOD) from wild tobacco (Nicotiana plumbaginifolia), Arabidopsis, and potato (Solanum tuberosum). Purified TRIP showed SOD activity, and Escherichia coli Fe-SOD was observed to have RIP activity too. Thus, TRIP may be considered a dual activity enzyme showing RIP-like activity and Fe-SOD characteristics.
Cyperus rotundus extract inhibits acetylcholinesterase activity from animal and plants as well as inhibits germination and seedling growth in wheat and tomato.

PubMed

Sharma, Rashmi; Gupta, Rajendra

2007-05-30

Cyperus rotundus (nutgrass) is the world's worst invasive weed through tubers. Its success in dominating natural habitats depends on its ability to prevent herbivory, and to kill or suppress other plants growing in its vicinity. The present study was done to investigate whether chemicals in nutgrass target neuronal and non-neuronal acetylcholinesterases to affect surrounding animals and plants respectively. Methanolic extract of tubers of nutgrass strongly inhibited activity of AChE from electric eel, wheat and tomato. It also inhibited seed germination and seedling growth in wheat and tomato. Our results suggest that inhibitor of AChE in nutgrass possibly acts as agent of plant's war against (a) herbivore animals, and (b) other plants trying to grow in the same habitat. An antiAChE from nutgrass has been purified by employing chromatography and crystallization. The structural determination of the purified inhibitor is in progress.
Studies on the enzymes of Sarcocystis suicanis: purification and characterization of an acid phosphatase.

PubMed

Farooqui, A A; Adams, D D; Hanson, W L; Prestwood, A K

1987-08-01

Percoll density gradient centrifugation was used for isolating large quantities of bradyzoites of Sarcocystis suicanis, which were used for enzymatic analysis. Crude extracts of bradyzoites contained activities suggestive of several acid hydrolases. Levels of acid and alkaline phosphatase were higher than those of beta-N-acetylhexosaminidase and beta-galactosidase. Acid phosphatase was purified 156-fold with an overall recovery of 54% using DEAE-Sepharose 4B and Sephadex G-200 chromatography. The partially purified enzyme was not a glycoprotein and had a molecular weight of approximately 170,000. The enzyme was markedly inhibited by Cu++, Hg++, and iodoacetamide, suggesting the presence of a sulfhydryl group. Sodium tartrate caused strong inhibition of the enzyme. The acid phosphatase of S. suicanis appears to be a unique enzyme that cannot be classified under high or low molecular weight acid phosphatases of widely diverse origin.
Liposomal Packaging Generates Wnt Protein with In Vivo Biological Activity

PubMed Central

Zhao, Ludan; Kim, Jae-Beom; ten Berge, Derk; Ponnusamy, Karthik; Carre, A. Lyonel; Dudek, Henryk; Zachlederova, Marie; McElhaney, Michael; Brunton, Shirley; Gunzner, Janet; Callow, Marinella; Polakis, Paul; Costa, Mike; Zhang, Xiaoyan M.; Helms, Jill A.; Nusse, Roel

2008-01-01

Wnt signals exercise strong cell-biological and regenerative effects of considerable therapeutic value. There are, however, no specific Wnt agonists and no method for in vivo delivery of purified Wnt proteins. Wnts contain lipid adducts that are required for activity and we exploited this lipophilicity by packaging purified Wnt3a protein into lipid vesicles. Rather than being encapsulated, Wnts are tethered to the liposomal surface, where they enhance and sustain Wnt signaling in vitro. Molecules that effectively antagonize soluble Wnt3a protein but are ineffective against the Wnt3a signal presented by a cell in a paracrine or autocrine manner are also unable to block liposomal Wnt3a activity, suggesting that liposomal packaging mimics the biological state of active Wnts. When delivered subcutaneously, Wnt3a liposomes induce hair follicle neogenesis, demonstrating their robust biological activity in a regenerative context. PMID:18698373
Liposomal packaging generates Wnt protein with in vivo biological activity.

PubMed

Morrell, Nathan T; Leucht, Philipp; Zhao, Ludan; Kim, Jae-Beom; ten Berge, Derk; Ponnusamy, Karthik; Carre, A Lyonel; Dudek, Henryk; Zachlederova, Marie; McElhaney, Michael; Brunton, Shirley; Gunzner, Janet; Callow, Marinella; Polakis, Paul; Costa, Mike; Zhang, Xiaoyan M; Helms, Jill A; Nusse, Roel

2008-08-13

Wnt signals exercise strong cell-biological and regenerative effects of considerable therapeutic value. There are, however, no specific Wnt agonists and no method for in vivo delivery of purified Wnt proteins. Wnts contain lipid adducts that are required for activity and we exploited this lipophilicity by packaging purified Wnt3a protein into lipid vesicles. Rather than being encapsulated, Wnts are tethered to the liposomal surface, where they enhance and sustain Wnt signaling in vitro. Molecules that effectively antagonize soluble Wnt3a protein but are ineffective against the Wnt3a signal presented by a cell in a paracrine or autocrine manner are also unable to block liposomal Wnt3a activity, suggesting that liposomal packaging mimics the biological state of active Wnts. When delivered subcutaneously, Wnt3a liposomes induce hair follicle neogenesis, demonstrating their robust biological activity in a regenerative context.
Pathogenesis-Related Proteins of Tomato 1

PubMed Central

Vera, Pablo; Conejero, Vicente

1988-01-01

An endoproteinase induced by citrus exocortis viroid has been purified from tomato (Lycopersicon esculentum Mill, cv “Rutgers”) leaves. The proteinase corresponds to one of the major pathogenesis-related proteins of tomato plants and was designated proteinase P-69 as it has a molecular weight of 69,000 to 70,000. The proteinase was purified in four steps: (NH4)2SO4 fractionation, chromatography on Bio-Gel P-60, DEAE-Sepharose chromatography, and casein-Sepharose affinity chromatography. The proteinase had a pH optimum of 8.5 to 9.0 when assayed with either fluorescein thiocarbamoyl derivative (FTC)-casein or FTC-ribulose 1,5-bisphosphate carboxylase/oxygenase as substrates. The proteinase activity was inhibited by pCMB and strongly activated by calcium and magnesium ions as well as by DTT. When analyzed by electrofocusing, the activity showed a pI around 9.0. Images Fig. 4 Fig. 8 PMID:16666127
Efficient renaturation of inclusion body proteins denatured by SDS.

PubMed

He, Chuan; Ohnishi, Kouhei

2017-09-02

Inclusion bodies are often formed when the foreign protein is over expressed in Escherichia coli. Since proteins in inclusion bodies are inactive, denaturing and refolding of inclusion body proteins are necessary to obtain the active form. Instead of the conventional denaturants, urea and guanidine hydrochloride, a strong anionic detergent SDS was used to solubilize C-terminal His-tag form of ulvan lyase in the inclusion bodies. Solution containing SDS-solubilized enzyme were kept on ice to precipitate SDS, followed by SDS-KCl insoluble crystal formation to remove SDS completely. After removing the precipitate by centrifugation, the supernatant was applied to Ni-NTA column to purify His-tagged ulvan lyase. The purified protein showed a dimeric form and ulvan lyase activity, demonstrating that SDS-denatured protein was renatured and recovered enzyme activity. This simple method could be useful for refolding other inclusion body proteins. Copyright © 2017 Elsevier Inc. All rights reserved.
Transcription factor MBF-I interacts with metal regulatory elements of higher eucaryotic metallothionein genes.

PubMed Central

Imbert, J; Zafarullah, M; Culotta, V C; Gedamu, L; Hamer, D

1989-01-01

Metallothionein (MT) gene promoters in higher eucaryotes contain multiple metal regulatory elements (MREs) that are responsible for the metal induction of MT gene transcription. We identified and purified to near homogeneity a 74-kilodalton mouse nuclear protein that specifically binds to certain MRE sequences. This protein, MBF-I, was purified employing as an affinity reagent a trout MRE that is shown to be functional in mouse cells but which lacks the G+C-rich and SP1-like sequences found in many mammalian MT gene promoters. Using point-mutated MREs, we showed that there is a strong correlation between DNA binding in vitro and MT gene regulation in vivo, suggesting a direct role of MBF-I in MT gene transcription. We also showed that MBF-I can induce MT gene transcription in vitro in a mouse extract and that this stimulation requires zinc. Images PMID:2586522
The Adsorption Capacity of GONs/CMC/Fe₃O₄ Magnetic Composite Microspheres and Applications for Purifying Dye Wastewater.

PubMed

Lv, Shenghua; Zhu, Linlin; Li, Ying; Jia, Chunmao; Sun, Shiyu

2017-01-11

Graphene oxide nanosheets (GONs)/carboxymethyl chitosan (CMC)/Fe₃O₄ magnetic composite microspheres (MCMs) were prepared by enclosing Fe₃O₄ particles with CMC and GONs in turn. The microstructures of GONs and GONs/CMC/Fe₃O₄ MCMs were characterized by FTIR, XRD, TEM, and SEM. The effects of GON content, pH value, and adsorption time on the adsorption capacity of the MCMs were investigated. The results show that the GONs/CMC/Fe₃O₄ MCMs have a greater specific surface area and a strong adsorption capacity for dye wastewater. Meanwhile, the adsorption mechanism was investigated, and the results accorded with the pseudo-second-order kinetic model and the Freundlich isotherm model. The search results indicate that GONs/CMC/Fe₃O₄ MCMs can be used to purify dye wastewater and has an important potential use in the practical purification of dye wastewater.
Structural analysis of an innate immunostimulant from broccoli, Brassica oleracea var. italica.

PubMed

Urai, Makoto; Kataoka, Keiko; Nishida, Satoshi; Sekimizu, Kazuhisa

2017-11-22

Vegetables are eaten as part of a healthy diet throughout the world, and some are also applied topically as a traditional medicine. We evaluated the innate immunostimulating activities of hot water extracts of various vegetables using the silkworm muscle contraction assay system, and found that broccoli, Brassica oleracea var. italica, contains a strong innate immunostimulant. We purified the innate immunostimulant from broccoli, and characterized the chemical structure by chemical analyses and NMR spectroscopy. The innate immunostimulant comprised galacturonic acid, galactose, glucose, arabinose, and rhamnose, and had a pectic-like polysaccharide structure. To determine the structural motif involved in the innate immunostimulating activity, we modified the structure by chemical and enzymatic treatment, and found that the activity was attenuated by pectinase digestion. These findings suggest that a pectic-like polysaccharide purified from broccoli has innate immune-stimulating activity, for which the polygalacturonic acid structure is necessary.
Purification and partial characterization of haloperoxidase from fresh water algae Cladophora glomerata.

PubMed

Verdel, E F; Kline, P C; Wani, S; Woods, A E

2000-02-01

Many haloperoxidases have been purified from diverse organisms, including lichen, fungi, bacteria, and marine algae. In this study a haloperoxidase was purified from the fresh water algae, Cladophora glomerata, by homogenization and centrifugation, ammonium sulfate fractionation, ion-exchange and gel filtration chromatography. Molecular weight was determined by SDS-PAGE and by size exclusion HPLC and found to be approximately 43 kDa. The isoelectric point was determined to be approximately 8.1 by isoelectric focusing. The UV spectrum of the peroxidase showed a strong absorbance in the Soret band indicating a heme protein, unlike vanadium-dependent haloperoxidases from marine algae. Fresh water algal haloperoxidase catalyzed the iodination of tyrosine at a pH of 3.1. This haloperoxidase also catalyzes the oxidation of guaiacol and oxidation of iodide as well as catalyzing a peroxide-dependent reaction in both the presence and absence of chloride and bromide ions.
Molecular adaptation and resilience of the insect’s nuclear receptor USP

PubMed Central

2012-01-01

Background The maintenance of biological systems requires plasticity and robustness. The function of the ecdysone receptor, a heterodimer composed of the nuclear receptors ECR (NR1H1) and USP (NR2B4), was maintained in insects despite a dramatic divergence that occurred during the emergence of Mecopterida. This receptor is therefore a good model to study the evolution of plasticity. We tested the hypothesis that selection has shaped the Ligand-Binding Domain (LBD) of USP during evolution of Mecopterida. Results We isolated usp and cox1 in several species of Drosophilidae, Tenebrionidae and Blattaria and estimated non-synonymous/synonymous rate ratios using maximum-likelihood methods and codon-based substitution models. Although the usp sequences were mainly under negative selection, we detected relaxation at residues located on the surface of the LBD within Mecopterida families. Using branch-site models, we also detected changes in selective constraints along three successive branches of the Mecopterida evolution. Residues located at the bottom of the ligand-binding pocket (LBP) underwent strong positive selection during the emergence of Mecopterida. This change is correlated with the acquisition of a large LBP filled by phospholipids that probably allowed the stabilisation of the new Mecopterida structure. Later, when the two subgroups of Mecopterida (Amphiesmenoptera: Lepidoptera, Trichoptera; Antliophora: Diptera, Mecoptera, Siphonaptera) diverged, the same positions became under purifying selection. Similarly, several positions of the heterodimerisation interface experienced positive selection during the emergence of Mecopterida, rapidly followed by a phase of constrained evolution. An enlargement of the heterodimerisation surface is specific for Mecopterida and was associated with a reinforcement of the obligatory partnership between ECR and USP, at the expense of homodimerisation. Conclusions In order to explain the episodic mode of evolution of USP, we propose a model in which the molecular adaptation of this protein is seen as a process of resilience for the maintenance of the ecdysone receptor functionality. PMID:23039844
Studies on the selective lysis and purification of Trypanosoma cruzi

PubMed Central

1975-01-01

The mechanism by which culture forms of Trypanosoma cruzi are lysed by normal mammalian sera was examined. Lysis is restricted to the epimastigote form of the organism and is not dependent on the presence of agglutinins. Lysis is a complement-dependent process, the activity being generated by the alternate pathway. The selective lysis by serum was exploited to purify viable trypomastigotes by means of centrifugation in an albumin column. Essentially pure trypomastigote populations are now being employed in cell culture experiments. PMID:807672
Retention and Molecular Evolution of Lipoxygenase Genes in Modern Rosid Plants

PubMed Central

Chen, Zhu; Chen, Danmei; Chu, Wenyuan; Zhu, Dongyue; Yan, Hanwei; Xiang, Yan

2016-01-01

Whole-genome duplication events have occurred more than once in the genomes of some rosids and played a significant role over evolutionary time. Lipoxygenases (LOXs) are involved in many developmental and resistance processes in plants. Our study concerns the subject of the LOX gene family; we tracked the evolutionary process of ancestral LOX genes in four modern rosids. Here we show that some members of the LOX gene family in the Arabidopsis genome are likely to be lost during evolution, leading to a smaller size than that in Populus, Vitis, and Carica. Strong purifying selection acted as a critical role in almost all of the paralogous and orthologous genes. The structure of LOX genes in Carica and Populus are relatively stable, whereas Vitis and Arabidopsis have a difference. By searching conserved motifs of LOX genes, we found that each sub-family shared similar components. Research on intraspecies gene collinearity show that recent duplication holds an important position in Populus and Arabidopsis. Gene collinearity analysis within and between these four rosid plants revealed that all LOX genes in each modern rosid were the offspring from different ancestral genes. This study traces the evolution of LOX genes which have been differentially retained and expanded in rosid plants. Our results presented here may aid in the selection of special genes retained in the rosid plants for further analysis of biological function. PMID:27746812
Sequencing and analysis of 10,967 full-length cDNA clones from Xenopus laevis and Xenopus tropicalis reveals post-tetraploidization transcriptome remodeling

PubMed Central

Morin, Ryan D.; Chang, Elbert; Petrescu, Anca; Liao, Nancy; Griffith, Malachi; Kirkpatrick, Robert; Butterfield, Yaron S.; Young, Alice C.; Stott, Jeffrey; Barber, Sarah; Babakaiff, Ryan; Dickson, Mark C.; Matsuo, Corey; Wong, David; Yang, George S.; Smailus, Duane E.; Wetherby, Keith D.; Kwong, Peggy N.; Grimwood, Jane; Brinkley, Charles P.; Brown-John, Mabel; Reddix-Dugue, Natalie D.; Mayo, Michael; Schmutz, Jeremy; Beland, Jaclyn; Park, Morgan; Gibson, Susan; Olson, Teika; Bouffard, Gerard G.; Tsai, Miranda; Featherstone, Ruth; Chand, Steve; Siddiqui, Asim S.; Jang, Wonhee; Lee, Ed; Klein, Steven L.; Blakesley, Robert W.; Zeeberg, Barry R.; Narasimhan, Sudarshan; Weinstein, John N.; Pennacchio, Christa Prange; Myers, Richard M.; Green, Eric D.; Wagner, Lukas; Gerhard, Daniela S.; Marra, Marco A.; Jones, Steven J.M.; Holt, Robert A.

2006-01-01

Sequencing of full-insert clones from full-length cDNA libraries from both Xenopus laevis and Xenopus tropicalis has been ongoing as part of the Xenopus Gene Collection Initiative. Here we present 10,967 full ORF verified cDNA clones (8049 from X. laevis and 2918 from X. tropicalis) as a community resource. Because the genome of X. laevis, but not X. tropicalis, has undergone allotetraploidization, comparison of coding sequences from these two clawed (pipid) frogs provides a unique angle for exploring the molecular evolution of duplicate genes. Within our clone set, we have identified 445 gene trios, each comprised of an allotetraploidization-derived X. laevis gene pair and their shared X. tropicalis ortholog. Pairwise dN/dS, comparisons within trios show strong evidence for purifying selection acting on all three members. However, dN/dS ratios between X. laevis gene pairs are elevated relative to their X. tropicalis ortholog. This difference is highly significant and indicates an overall relaxation of selective pressures on duplicated gene pairs. We have found that the paralogs that have been lost since the tetraploidization event are enriched for several molecular functions, but have found no such enrichment in the extant paralogs. Approximately 14% of the paralogous pairs analyzed here also show differential expression indicative of subfunctionalization. PMID:16672307
Comparative Genomics Reveals Accelerated Evolution in Conserved Pathways during the Diversification of Anole Lizards.

PubMed

Tollis, Marc; Hutchins, Elizabeth D; Stapley, Jessica; Rupp, Shawn M; Eckalbar, Walter L; Maayan, Inbar; Lasku, Eris; Infante, Carlos R; Dennis, Stuart R; Robertson, Joel A; May, Catherine M; Crusoe, Michael R; Bermingham, Eldredge; DeNardo, Dale F; Hsieh, Shi-Tong Tonia; Kulathinal, Rob J; McMillan, William Owen; Menke, Douglas B; Pratt, Stephen C; Rawls, Jeffery Alan; Sanjur, Oris; Wilson-Rawls, Jeanne; Wilson Sayres, Melissa A; Fisher, Rebecca E; Kusumi, Kenro

2018-02-01

Squamates include all lizards and snakes, and display some of the most diverse and extreme morphological adaptations among vertebrates. However, compared with birds and mammals, relatively few resources exist for comparative genomic analyses of squamates, hampering efforts to understand the molecular bases of phenotypic diversification in such a speciose clade. In particular, the ∼400 species of anole lizard represent an extensive squamate radiation. Here, we sequence and assemble the draft genomes of three anole species-Anolis frenatus, Anolis auratus, and Anolis apletophallus-for comparison with the available reference genome of Anolis carolinensis. Comparative analyses reveal a rapid background rate of molecular evolution consistent with a model of punctuated equilibrium, and strong purifying selection on functional genomic elements in anoles. We find evidence for accelerated evolution in genes involved in behavior, sensory perception, and reproduction, as well as in genes regulating limb bud development and hindlimb specification. Morphometric analyses of anole fore and hindlimbs corroborated these findings. We detect signatures of positive selection across several genes related to the development and regulation of the forebrain, hormones, and the iguanian lizard dewlap, suggesting molecular changes underlying behavioral adaptations known to reinforce species boundaries were a key component in the diversification of anole lizards. © The Author(s) 2018. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.
Amylase production by Preussia minima, a fungus of endophytic origin: optimization of fermentation conditions and analysis of fungal secretome by LC-MS

PubMed Central

2014-01-01

Background Environmental screening programs are used to find new enzymes that may be utilized in large-scale industrial processes. Among microbial sources of new enzymes, the rationale for screening fungal endophytes as a potential source of such enzymes relates to the hypothesised mutualistic relationship between the endophyte and its host plant. There is a need for new microbial amylases that are active at low temperature and alkaline conditions as these would find industrial applications as detergents. Results An α-amylase produced by Preussia minima, isolated from the Australian native plant, Eremophilia longifolia, was purified to homogeneity through fractional acetone precipitation and Sephadex G-200 gel filtration, followed by DEAE-Sepharose ion exchange chromatography. The purified α-amylase showed a molecular mass of 70 kDa which was confirmed by zymography. Temperature and pH optima were 25°C and pH 9, respectively. The enzyme was activated and stabilized mainly by the metal ions manganese and calcium. Enzyme activity was also studied using different carbon and nitrogen sources. It was observed that enzyme activity was highest (138 U/mg) with starch as the carbon source and L-asparagine as the nitrogen source. Bioreactor studies showed that enzyme activity was comparable to that obtained in shaker cultures, which encourages scale-up fermentation for enzyme production. Following in-gel digestion of the purified protein by trypsin, a 9-mer peptide was sequenced and analysed by LC-ESI-MS/MS. The partial amino acid sequence of the purified enzyme presented similarity to α-amylase from Magnaporthe oryzae. Conclusions The findings of the present study indicate that the purified α-amylase exhibits a number of promising properties that make it a strong candidate for application in the detergent industry. To our knowledge, this is the first amylase isolated from a Preussia minima strain of endophytic origin. PMID:24602289
Amylase production by Preussia minima, a fungus of endophytic origin: optimization of fermentation conditions and analysis of fungal secretome by LC-MS.

PubMed

Zaferanloo, Bita; Bhattacharjee, Shatabdi; Ghorbani, Mahmood M; Mahon, Peter J; Palombo, Enzo A

2014-03-07

Environmental screening programs are used to find new enzymes that may be utilized in large-scale industrial processes. Among microbial sources of new enzymes, the rationale for screening fungal endophytes as a potential source of such enzymes relates to the hypothesised mutualistic relationship between the endophyte and its host plant. There is a need for new microbial amylases that are active at low temperature and alkaline conditions as these would find industrial applications as detergents. An α-amylase produced by Preussia minima, isolated from the Australian native plant, Eremophilia longifolia, was purified to homogeneity through fractional acetone precipitation and Sephadex G-200 gel filtration, followed by DEAE-Sepharose ion exchange chromatography. The purified α-amylase showed a molecular mass of 70 kDa which was confirmed by zymography. Temperature and pH optima were 25°C and pH 9, respectively. The enzyme was activated and stabilized mainly by the metal ions manganese and calcium. Enzyme activity was also studied using different carbon and nitrogen sources. It was observed that enzyme activity was highest (138 U/mg) with starch as the carbon source and L-asparagine as the nitrogen source. Bioreactor studies showed that enzyme activity was comparable to that obtained in shaker cultures, which encourages scale-up fermentation for enzyme production. Following in-gel digestion of the purified protein by trypsin, a 9-mer peptide was sequenced and analysed by LC-ESI-MS/MS. The partial amino acid sequence of the purified enzyme presented similarity to α-amylase from Magnaporthe oryzae. The findings of the present study indicate that the purified α-amylase exhibits a number of promising properties that make it a strong candidate for application in the detergent industry. To our knowledge, this is the first amylase isolated from a Preussia minima strain of endophytic origin.

In vitro study of histamine and histamine receptor ligands influence on the adhesion of purified human eosinophils to endothelium.

PubMed

Grosicki, Marek; Wójcik, Tomasz; Chlopicki, Stefan; Kieć-Kononowicz, Katarzyna

2016-04-15

It is a well-known fact that histamine is involved in eosinophil-dependent inflammatory responses including cellular chemotaxis and migration. Nevertheless, the relative role of histamine receptors in the mechanisms of eosinophils adhesion to endothelial cells is not known. Therefore the aim of presented study was to examine the effect of selective histamine receptors ligands on eosinophils adhesion to endothelium. For that purpose the highly purified human eosinophils have been isolated from the peripheral blood. The viability and functional integrity of isolated eosinophils have been validated in several tests. Histamine as well as 4-methylhistamine (selective H4 agonist) in concentration-dependent manner significantly increased number of eosinophils that adhere to endothelium. Among the selective histamine receptors antagonist or H1 inverse agonist only JNJ7777120 (histamine H4 antagonist) and thioperamide (dual histamine H3/H4 antagonist) had direct effect on eosinophils adhesion to endothelial cells. Antagonists of H1 (diphenhydramine, mepyramine) H2 (ranitidine and famotidine) and H3 (pitolisant) histamine receptors were ineffective. To the best of our knowledge, this is the first study to demonstrate that histamine receptor H4 plays a dominant role in histamine-induced eosinophils adhesion to endothelium. Copyright © 2016 Elsevier B.V. All rights reserved.
The NS3 proteins of global strains of bluetongue virus evolve into regional topotypes through negative (purifying) selection.

PubMed

Balasuriya, U B R; Nadler, S A; Wilson, W C; Pritchard, L I; Smythe, A B; Savini, G; Monaco, F; De Santis, P; Zhang, N; Tabachnick, W J; Maclachlan, N J

2008-01-01

Comparison of the deduced amino acid sequences of the genes (S10) encoding the NS3 protein of 137 strains of bluetongue virus (BTV) from Africa, the Americas, Asia, Australia and the Mediterranean Basin showed limited variation. Common to all NS3 sequences were potential glycosylation sites at amino acid residues 63 and 150 and a cysteine at residue 137, whereas a cysteine at residue 181 was not conserved. The PPXY and PS/TAP late-domain motifs were conserved in all but three of the viruses. Phylogenetic analyses of these same sequences yielded two principal clades that grouped the viruses irrespective of their serotype or year of isolation (1900-2003). All viruses from Asia and Australia were grouped in one clade, whereas those from the other regions were present in both clades. Each clade segregated into distinct subclades that included viruses from single or multiple regions, and the S10 genes of some field viruses were identical to those of live-attenuated BTV vaccines. There was no evidence of positive selection on the S10 gene as assessed by reconstruction of ancestral codon states on the phylogeny, rather the functional constraints of the NS3 protein are expressed through substantial negative (purifying) selection.
Purification of bioactive phenolics from Phanerochaete chysosporium biomass extract on selected macroporous resins

NASA Astrophysics Data System (ADS)

Idris, Z. M.; Dzahir, M. I. H. M.; Jamal, P.; Barkat, A. A.; Xian, R. L. W.

2017-06-01

In this study, two different types of macroporous resins known as XAD-7HP and HP-20 were evaluated for the adsorption and desorption properties against bioactive phenolics extracted from Phanerochaete chrysosporium. From the previous static sorption studies, it was found that the adsorption capacity for both resins had has no significant difference. Then, the kinetic adsorption data were analyzed with both pseudo-first-order and pseudo-second-order equations and the later performed better. The adsorption isotherm data were fitted well by both Langmuir and Freundlich models. Meanwhile in desorption study, HP-20 and XAD-7HP gave 90.52% and 88.28% recoveries, respectively. Considering the desorption results of the macroporous resins, HP-20 and XAD-7HP were packed in chromatography column to further purify the phenolics. For dynamic adsorption, breakthrough capacity of HP-20 (0.522) was found to be higher than XAD-7HP (0.131). Different ethanol concentrations (30% to 50% (v/v)) were investigated at fixed flowrate (1 ml/min) on phenolics recovery from both types of resins. The highest recovery of bioactive phenolics was 94.3% using XAD-7HP resins at 50% (v/v) of ethanol. Only 77.1% of bioactive phenolics were recovered using HP-20 resin at the same experimental conditions. The purified extract subsequently was analyzed using HPLC. The results showed that three phenolics (gallic acid 3,4-dihydroxybenzoic acid and 4-hydroxybenzoic acid) were identified with higher concentrations as compared to non-purified extract. Finally, the purified extract was tested for scavenging activity against DPPH, and it showed that the activity increased significantly to 90.80% from 59.94% in non-purified extract.
Method for removal of phosgene from boron trichloride

DOEpatents

Freund, S.M.

1983-09-20

Selective ultraviolet photolysis using an unfiltered mercury arc lamp has been used to substantially reduce the phosgene impurity in a mixture of boron trichloride and phosgene. Infrared spectrophotometric analysis of the sample before and after irradiation shows that it is possible to highly purify commercially available boron trichloride with this method. 5 figs.
ADENOVIRUS INTERACTION WITH ITS CELLULAR RECEPTOR CAR.

DOE Office of Scientific and Technical Information (OSTI.GOV)

HOWITT,J.; ANDERSON,C.W.; FREIMUTH,P.

The mechanism of adenovirus attachment to the host cell plasma membrane has been revealed in detail by research over the past 10 years. It has long been known that receptor binding activity is associated with the viral fibers, trimeric spike proteins that protrude radially from the vertices of the icosahedral capsid (Philipson et al. 1968). In some adenovirus serotypes, fiber and other virus structural proteins are synthesized in excess and accumulate in the cell nucleus during late stages of infection. Fiber protein can be readily purified from lysates of cells infected with subgroup C viruses, for example Ad2 and Ad5more » (Boulanger and Puvion 1973). Addition of purified fiber protein to virus suspensions during adsorption strongly inhibits infection, indicating that fiber and intact virus particles compete for binding sites on host cells (Philipson et al. 1968; Hautala et al. 1998). Cell binding studies using purified radiolabeled fiber demonstrated that fiber binds specifically and with high affinity to the cell plasma membrane, and that cell lines typically used for laboratory propagation of adenovirus have approximately 10{sup 4} high-affinity receptor sites per cell (Persson et al. 1985; Freimuth 1996). Similar numbers of high-affinity binding sites for radiolabeled intact virus particles also were observed (Seth et al. 1994).« less
Purification, characterization, and crystallization of Crocodylus siamensis hemoglobin.

PubMed

Jandaruang, Jinda; Siritapetawee, Jaruwan; Songsiriritthigul, Chomphunuch; Preecharram, Sutthidech; Azuma, Taoka; Dhiravisit, Apisak; Fukumori, Yoshihiro; Thammasirirak, Sompong

2014-08-01

Crocodylus siamensis hemoglobin was purified by a size exclusion chromatography, Sephacryl S-100 with buffer containing dithiothreitol. The purified Hb was dissociated to be two forms (α chain and β chain) which observed by SDS-PAGE, indicated that the C. siamensis Hb was an unpolymerized form. The unpolymerized Hb (composed of two α chains and two β chains) showed high oxygen affinity at 3.13 mmHg (P(50)) and 1.96 (n value), and a small Bohr effect (δH(+) = -0.29) at a pH of 6.9-8.4. Adenosine triphosphate did not affect the oxygenation properties, whereas bicarbonate ions strongly depressed oxygen affinity. Crude C. siamensis Hb solutions were showed high O(2) affinity at P(50) of 2.5 mmHg which may assure efficient utilization of the lung O(2) reserve during breath holding and diving. The purified Hbs were changed to cyanmethemoglobin forms prior crystallization. Rod- and plate-shaped crystals were obtained by the sitting-drop vapor-diffusion method at 5 °C using equal volumes of protein solution (37 mg/ml) and reservoir [10-13 % (w/v) PEG 4000, with 0.1 M Tris buffer in present of 0.2 M MgCl(2)·6H(2)O] solution at a pH of 7.0-8.5.
Optimization, purification, and characterization of L-asparaginase from Actinomycetales bacterium BkSoiiA.

PubMed

Dash, Chitrangada; Mohapatra, Sukanti Bala; Maiti, Prasanta Kumar

2016-01-01

Actinobacteria are promising source of a wide range of important enzymes, some of which are produced in industrial scale, with others yet to be harnessed. L-Asparaginase is used as an antineoplastic agent. The present work deals with the production and optimization of L-asparaginase from Actinomycetales bacterium BkSoiiA using submerged fermentation in M9 medium. Production optimization resulted in a modified M9 medium with yeast extract and fructose as carbon and nitrogen sources, respectively, at pH 8.0, incubated for 120 hr at 30 ± 2 °C. The crude enzyme was purified to near homogeneity by ammonium sulfate precipitation following dialysis, ion-exchange column chromatography, and finally gel filtration. The sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) revealed an apparent molecular weight of 57 kD. The enzyme was purified 95.06-fold and showed a final specific activity of 204.37 U/mg with 3.49% yield. The purified enzyme showed maximum activity at a pH 10.0 and was stable at pH 7.0 to 9.0. The enzyme was activated by Mn(2+) and strongly inhibited by Ba(2+). All these preliminary characterization suggests that the L-asparaginase from the source may be a tool useful to pharmaceutical industries after further research.
Identification and structural characterization of a new pro-apoptotic cyclic octapeptide cyclosaplin from somatic seedlings of Santalum album L.

PubMed

Mishra, Abheepsa; Gauri, Samiran S; Mukhopadhyay, Sourav K; Chatterjee, Soumya; Das, Shibendu S; Mandal, Santi M; Dey, Satyahari

2014-04-01

Small cyclic peptides exhibiting potent biological activity have great potential for anticancer therapy. An antiproliferative cyclic octapeptide, cyclosaplin was purified from somatic seedlings of Santalum album L. (sandalwood) using gel filtration and RP-HPLC separation process. The molecular mass of purified peptide was found to be 858 Da and the sequence was determined by MALDI-ToF-PSD-MS as 'RLGDGCTR' (cyclic). The cytotoxic activity of the peptide was tested against human breast cancer (MDA-MB-231) cell line in a dose and time-dependent manner. The purified peptide exhibited significant antiproliferative activity with an IC50 2.06 μg/mL. In a mechanistic approach, apoptosis was observed in differential microscopic studies for peptide treated MDA-MB-231 cells, which was further confirmed by mitochondrial membrane potential, DNA fragmentation assay, cell cycle analysis and caspase 3 activities. The modeling and docking experiments revealed strong affinity (kcal/mol) of peptide toward EGFR and procaspase 3. The co-localization studies revealed that the peptide sensitizes MDA-MB-231 cells by possibly binding to EGFR and induces apoptosis. This unique cyclic octapeptide revealed to be a favorable candidate for development of anticancer agents. Copyright © 2014 Elsevier Inc. All rights reserved.
Improved soluble expression and characterization of the Hc domain of Clostridium botulinum neurotoxin serotype A in Escherichia coli by using a PCR-synthesized gene and a Trx co-expression strain.

PubMed

Chen, Rongchang; Shi, Jing; Cai, Kun; Tu, Wei; Hou, Xiaojun; Liu, Hao; Xiao, Le; Wang, Qin; Tang, Yunming; Wang, Hui

2010-05-01

Botulinum neurotoxin serotype A (BoNT/A) is an extremely potent bacterial protein toxin. The Hc fragment of BoNT/A (AHc) was shown to be non-toxic, antigenic, and capable of eliciting a protective immunity in animals challenged with homologous BoNT. In this study, we synthesized AHc gene by using T4 DNA ligase and PCR. The AHc was expressed at a high level in Escherichia coli successfully. Because of using the Trx co-expression strain, the expressed AHc is in a soluble and active form. The yield of the purified AHc was about 70mg/L, and its purity was up to 90% through one-step affinity chromatography. The AHc was positively identified by the antibodies raised against BoNT/A using immunological-dot-blot and Western blot assays. AHc was shown to bind with gangliosides and elicit immunity against BoNT/A, indicating that the expressed and purified AHc protein retains a functionally active conformation. Furthermore, the purified AHc has a strong immunogenicity and can be used as a potential subunit candidate vaccine for botulinum toxin serotype A. Copyright (c) 2009 Elsevier Inc. All rights reserved.
Kluyveromyces wickerhamii killer toxin: purification and activity towards Brettanomyces/Dekkera yeasts in grape must.

PubMed

Comitini, Francesca; Ciani, Maurizio

2011-03-01

Brettanomyces/Dekkera yeasts have been identified as part of the grape yeast flora. They are well known for colonizing the cellar environmental and spoiling wines, causing haze, turbidity and strong off-flavours in wines and enhancing the volatile acidity. As the general practices applied to combat Brettanomyces/Dekkera yeasts are not particularly appropriate during wine ageing and storage, a biological alternative to curtailing their growth would be welcomed in winemaking. In this study, we investigated the Kluyveromyces wickerhamii killer toxin (Kwkt) that is active against Brettanomyces/Dekkera spoilage yeasts. Purification procedures allowed the identification of Kwkt as a protein with an apparent molecular mass of 72 kDa and without any glycosyl residue. Interestingly, purified Kwkt has fungicidal effects at low concentrations under the physicochemical conditions of winemaking. The addition of 40 and 80 mg L(-1) purified Kwkt showed efficient antispoilage effects, controlling both growth and metabolic activity of sensitive spoilage yeasts. At these two killer toxin concentrations, compounds known to contribute to the 'Brett' character of wines, such as ethyl phenols, were not produced. Thus, purified Kwkt appears to be a suitable biological strategy to control Brettanomyces/Dekkera yeasts during fermentation, wine ageing and storage. © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.
Adaptation of the Haloarcula hispanica CRISPR-Cas system to a purified virus strictly requires a priming process

PubMed Central

Li, Ming; Wang, Rui; Zhao, Dahe; Xiang, Hua

2014-01-01

The clustered regularly interspaced short palindromic repeat (CRISPR)-Cas system mediates adaptive immunity against foreign nucleic acids in prokaryotes. However, efficient adaptation of a native CRISPR to purified viruses has only been observed for the type II-A system from a Streptococcus thermophilus industry strain, and rarely reported for laboratory strains. Here, we provide a second native system showing efficient adaptation. Infected by a newly isolated virus HHPV-2, Haloarcula hispanica type I-B CRISPR system acquired spacers discriminatively from viral sequences. Unexpectedly, in addition to Cas1, Cas2 and Cas4, this process also requires Cas3 and at least partial Cascade proteins, which are involved in interference and/or CRISPR RNA maturation. Intriguingly, a preexisting spacer partially matching a viral sequence is also required, and spacer acquisition from upstream and downstream sequences of its target sequence (i.e. priming protospacer) shows different strand bias. These evidences strongly indicate that adaptation in this system strictly requires a priming process. This requirement, if validated also true for other CRISPR systems as implied by our bioinformatic analysis, may help to explain failures to observe efficient adaptation to purified viruses in many laboratory strains, and the discrimination mechanism at the adaptation level that has confused scientists for years. PMID:24265226
Purification and partial characterization of a new mannose/glucose-specific lectin from Dialium guineense Willd seeds that exhibits toxic effect.

PubMed

Bari, Alfa U; Silva, Helton C; Silva, Mayara T L; Pereira Júnior, Francisco N; Cajazeiras, João B; Sampaio, Alexandre H; Leal, Rodrigo B; Teixeira, Edson H; Rocha, Bruno A M; Nascimento, Kyria S; Nagano, Celso S; Cavada, Benildo S

2013-08-01

A new mannose/glucose-specific lectin, named DigL, was purified from seeds of Dialium guineense by a single step using a Sepharose 4b-Mannose affinity chromatography column. DigL strongly agglutinated rabbit erythrocytes and was inhibited by d-mannose, d-glucose, and derived sugars, especially α-methyl-d-mannopyranoside and N-acetyl-d-glucosamine. DigL has been shown to be a stable protein, maintaining its hemagglutinating activity after incubation at a wide range of temperature and pH values and after incubation with EDTA. DigL is a glycoprotein composite by approximately 2.9% of carbohydrates by weight. By sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis, the purified DigL exhibited an electrophoretic profile consisting of a broad band of 28-30 kDa. Analysis using electrospray ionization mass spectrometry indicated that purified DigL possesses a molecular average mass of 28 452 ± 2 Da and shows the presence of possible glycoforms. In addition, DigL exhibited an intermediary toxic effect on Artemia sp. nauplii, and this effect was both dependent on native structure and mediated by a carbohydrate-binding site. Copyright © 2013 John Wiley & Sons, Ltd.
Fractionation and Purification of Bioactive Compounds Obtained from a Brewery Waste Stream

PubMed Central

Barbosa-Pereira, Letricia; Pocheville, Ainara; Angulo, Inmaculada; Paseiro-Losada, Perfecto; Cruz, Jose M.

2013-01-01

The brewery industry generates waste that could be used to yield a natural extract containing bioactive phenolic compounds. We compared two methods of purifying the crude extract—solid-phase extraction (SPE) and supercritical fluid extraction (SFE)—with the aim of improving the quality of the final extract for potential use as safe food additive, functional food ingredient, or nutraceutical. The predominant fractions yielded by SPE were the most active, and the fraction eluted with 30% (v/v) of methanol displayed the highest antioxidant activity (0.20 g L−1), similar to that of BHA. The most active fraction yielded by SFE (EC50 of 0.23 g L−1) was obtained under the following conditions: temperature 40°C, pressure 140 bar, extraction time 30 minutes, ethanol (6%) as a modifier, and modifier flow 0.2 mL min−1. Finally, we found that SFE is the most suitable procedure for purifying the crude extracts and improves the organoleptic characteristics of the product: the final extract was odourless, did not contain solvent residues, and was not strongly coloured. Therefore, natural extracts obtained from the residual stream and purified by SFE can be used as natural antioxidants with potential applications in the food, cosmetic, and pharmaceutical industries. PMID:23762844
An efficient process for obtaining prebiotic oligosaccharides derived from lactulose using isomerized and purified whey permeate.

PubMed

Sabater, Carlos; Olano, Agustín; Prodanov, Marin; Montilla, Antonia; Corzo, Nieves

2017-12-01

One of the most promising uses of whey permeate (WP) is the synthesis of prebiotic oligosaccharides. Herein, commercial WP was submitted to chemical isomerization catalysed by sodium borate at an alkaline pH and subsequent purification using anion-exchange resins to remove boron. Subsequently, purified mixtures were used to synthesize prebiotic oligosaccharides using β-galactosidase from Bacillus circulans. Isomerization of concentrated WP (200 g L -1 lactose) gave rise to levels of lactulose up to 155.5 g L -1 after 30 min of reaction (molar ratio of boron/lactose, 1/1; pH 12; 70 °C). Boron was removed from the isomerized WP (IWP) using the combination of a strong acid (IR-120, H + ) and a weak base (IRA-743) anion-exchange resins, reducing its level to <1 ppm, without loss of lactulose. During the transglycosylation reaction of purified IWP (lactose/lactulose ratio, 1/2.4) maximum content of prebiotic compounds was achieved, i.e. 690 g kg -1 WP after 3 h of reaction. This study shows that combined chemical-enzymatic reactions together with the purification of IWP results in an efficient synthesis of prebiotic oligosaccharides. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.
Tempo and Mode in the Molecular Evolution of Influenza C

PubMed Central

Gatherer, Derek

2010-01-01

Abstract: Influenza C contributes to economic damage caused by working days lost through absence or inefficiency and may occasionally cause an acute respiratory illness in a paediatric setting. All Influenza C sequences from the NCBI Influenza Virus Resource were examined to determine the date of the most recent common ancestor (t-MRCA), the average nucleotide substitution rate, and the location of residues under positive selection, for each of the seven genome segments of this virus. The segment with the deepest phylogeny was found to be segment 4, encoding the haemagglutinin-esterase protein (HE) with mean t-MRCA at 1890 of the common era (AD), at a 95% highest posterior density (HPD) of 1857-1924 AD. Other genome segments have slightly more recent common ancestors, ranging from mean t-MRCAs of 1916 AD (HPD 1891-1937) for segment 7, encoding the two non-structural proteins (NS) to 1944 AD (HPD 1940-1948) for segment 2 encoding the type 1 basic polymerase (PB1). On the basis of the Bayesian analysis a reclassification of lineages within genome segments is proposed. Some evidence for positive selection was found in the receptor-binding domain of the haemagglutinin-esterase protein. However, average ω (omega) values ranged from 0.05 for polymerase basic protein 2 (PB2) to 0.38 for non-structural protein 2 (NS2), suggesting that strong to moderate purifying selection is the main trend. Characteristic combinations of segment lineages were identified (genome constellations) and shown to have a relatively short life-span before being broken up by reassortment. PMID:21127722
Interactions of the sulfonylurea receptor 1 subunit in the molecular assembly of beta-cell K(ATP) channels.

PubMed

Mikhailov, M V; Ashcroft, S J

2000-02-04

We have investigated protein interactions involved in pancreatic beta-cell ATP-sensitive potassium channel assembly. These channels, which are of key importance for control of insulin release, are a hetero-oligomeric complex of pore-forming Kir6.2 subunits and sulfonylurea receptor (SUR1) subunits with two nucleotide-binding domains (NBD1 and NBD2). We divided SUR1 into two halves at Pro-1042. Expression of either the individual N- or C-terminal domain in a baculovirus expression system did not lead to glibenclamide binding activity, although studies with green fluorescent protein fusion proteins showed that both half-molecules were inserted into the plasma membrane. However, significant glibenclamide binding activity was observed when the half-molecules were co-expressed (even when NBD2 was deleted from the C-terminal half-molecule). Simultaneous expression of Kir6.2 resulted in enhanced glibenclamide binding activity. We conclude that the glibenclamide-binding site includes amino acid residues from both halves of the molecule, that there is strong interaction between different regions of SUR1, that NBD2 is not essential for glibenclamide binding, and that interactions between Kir6.2 and SUR1 participate in ATP-sensitive potassium channel assembly. Investigation of NBD1-green fluorescent protein fusion protein distribution inside insect cells expressing C-terminal halves of SUR1 demonstrated strong interaction between NBD1 and NBD2. We also expressed and purified NBD1 from Escherichia coli. Purified NBD1 was found to exist as a tetramer indicating strong homomeric attractions and a possible role for NBD1 in SUR1 assembly.
Genomic analysis reveals major determinants of cis-regulatory variation in Capsella grandiflora

PubMed Central

Steige, Kim A.; Laenen, Benjamin; Reimegård, Johan; Slotte, Tanja

2017-01-01

Understanding the causes of cis-regulatory variation is a long-standing aim in evolutionary biology. Although cis-regulatory variation has long been considered important for adaptation, we still have a limited understanding of the selective importance and genomic determinants of standing cis-regulatory variation. To address these questions, we studied the prevalence, genomic determinants, and selective forces shaping cis-regulatory variation in the outcrossing plant Capsella grandiflora. We first identified a set of 1,010 genes with common cis-regulatory variation using analyses of allele-specific expression (ASE). Population genomic analyses of whole-genome sequences from 32 individuals showed that genes with common cis-regulatory variation (i) are under weaker purifying selection and (ii) undergo less frequent positive selection than other genes. We further identified genomic determinants of cis-regulatory variation. Gene body methylation (gbM) was a major factor constraining cis-regulatory variation, whereas presence of nearby transposable elements (TEs) and tissue specificity of expression increased the odds of ASE. Our results suggest that most common cis-regulatory variation in C. grandiflora is under weak purifying selection, and that gene-specific functional constraints are more important for the maintenance of cis-regulatory variation than genome-scale variation in the intensity of selection. Our results agree with previous findings that suggest TE silencing affects nearby gene expression, and provide evidence for a link between gbM and cis-regulatory constraint, possibly reflecting greater dosage sensitivity of body-methylated genes. Given the extensive conservation of gbM in flowering plants, this suggests that gbM could be an important predictor of cis-regulatory variation in a wide range of plant species. PMID:28096395
Selection for low dormancy in annual ryegrass (Lolium rigidum) seeds results in high constitutive expression of a glucose-responsive α-amylase isoform

PubMed Central

Goggin, Danica E.; Powles, Stephen B.

2012-01-01

Background and Aims α-Amylase in grass caryopses (seeds) is usually expressed upon commencement of germination and is rarely seen in dry, mature seeds. A heat-stable α-amylase activity was unexpectedly selected for expression in dry annual ryegrass (Lolium rigidum) seeds during targeted selection for low primary dormancy. The aim of this study was to characterize this constitutive activity biochemically and determine if its presence conferred insensitivity to the germination inhibitors abscisic acid and benzoxazolinone. Methods α-Amylase activity in developing, mature and germinating seeds from the selected (low-dormancy) and a field-collected (dormant) population was characterized by native activity PAGE. The response of seed germination and α-amylase activity to abscisic acid and benzoxazolinone was assessed. Using an alginate affinity matrix, α-amylase was purified from dry and germinating seeds for analysis of its enzymatic properties. Key Results The constitutive α-amylase activity appeared late during seed development and was mainly localized in the aleurone; in germinating seeds, this activity was responsive to both glucose and gibberellin. It migrated differently on native PAGE compared with the major activities in germinating seeds of the dormant population, but the enzymatic properties of α-amylase purified from the low-dormancy and dormant seeds were largely indistinguishable. Seed imbibition on benzoxazolinone had little effect on the low-dormancy seeds but greatly inhibited germination and α-amylase activity in the dormant population. Conclusions The constitutive α-amylase activity in annual ryegrass seeds selected for low dormancy is electrophoretically different from that in germinating seeds and its presence confers insensitivity to benzoxazolinone. The concurrent selection of low dormancy and constitutive α-amylase activity may help to enhance seedling establishment under competitive conditions. PMID:23002268
Serum Albumin Domain Structures in Human Blood Serum by Mass Spectrometry and Computational Biology.

PubMed

Belsom, Adam; Schneider, Michael; Fischer, Lutz; Brock, Oliver; Rappsilber, Juri

2016-03-01

Chemical cross-linking combined with mass spectrometry has proven useful for studying protein-protein interactions and protein structure, however the low density of cross-link data has so far precluded its use in determining structures de novo. Cross-linking density has been typically limited by the chemical selectivity of the standard cross-linking reagents that are commonly used for protein cross-linking. We have implemented the use of a heterobifunctional cross-linking reagent, sulfosuccinimidyl 4,4'-azipentanoate (sulfo-SDA), combining a traditional sulfo-N-hydroxysuccinimide (sulfo-NHS) ester and a UV photoactivatable diazirine group. This diazirine yields a highly reactive and promiscuous carbene species, the net result being a greatly increased number of cross-links compared with homobifunctional, NHS-based cross-linkers. We present a novel methodology that combines the use of this high density photo-cross-linking data with conformational space search to investigate the structure of human serum albumin domains, from purified samples, and in its native environment, human blood serum. Our approach is able to determine human serum albumin domain structures with good accuracy: root-mean-square deviation to crystal structure are 2.8/5.6/2.9 Å (purified samples) and 4.5/5.9/4.8Å (serum samples) for domains A/B/C for the first selected structure; 2.5/4.9/2.9 Å (purified samples) and 3.5/5.2/3.8 Å (serum samples) for the best out of top five selected structures. Our proof-of-concept study on human serum albumin demonstrates initial potential of our approach for determining the structures of more proteins in the complex biological contexts in which they function and which they may require for correct folding. Data are available via ProteomeXchange with identifier PXD001692. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.
Stubborn contaminants: influence of detergents on the purity of the multidrug ABC transporter BmrA.

PubMed

Wiseman, Benjamin; Kilburg, Arnaud; Chaptal, Vincent; Reyes-Mejia, Gina Catalina; Sarwan, Jonathan; Falson, Pierre; Jault, Jean-Michel

2014-01-01

Despite the growing interest in membrane proteins, their crystallization remains a major challenge. In the course of a crystallographic study on the multidrug ATP-binding cassette transporter BmrA, mass spectral analyses on samples purified with six selected detergents revealed unexpected protein contamination visible for the most part on overloaded SDS-PAGE. A major contamination from the outer membrane protein OmpF was detected in purifications with Foscholine 12 (FC12) but not with Lauryldimethylamine-N-oxide (LDAO) or any of the maltose-based detergents. Consequently, in the FC12 purified BmrA, OmpF easily crystallized over BmrA in a new space group, and whose structure is reported here. We therefore devised an optimized protocol to eliminate OmpF during the FC12 purification of BmrA. On the other hand, an additional band visible at ∼110 kDa was detected in all samples purified with the maltose-based detergents. It contained AcrB that crystallized over BmrA despite its trace amounts. Highly pure BmrA preparations could be obtained using either a ΔacrAB E. coli strain and n-dodecyl-β-D-maltopyranoside, or a classical E. coli strain and lauryl maltose neopentyl glycol for the overexpression and purification, respectively. Overall our results urge to incorporate a proteomics-based purity analysis into quality control checks prior to commencing crystallization assays of membrane proteins that are notoriously arduous to crystallize. Moreover, the strategies developed here to selectively eliminate obstinate contaminants should be applicable to the purification of other membrane proteins overexpressed in E. coli.

Stubborn Contaminants: Influence of Detergents on the Purity of the Multidrug ABC Transporter BmrA

PubMed Central

Chaptal, Vincent; Reyes-Mejia, Gina Catalina; Sarwan, Jonathan; Falson, Pierre; Jault, Jean-Michel

2014-01-01

Despite the growing interest in membrane proteins, their crystallization remains a major challenge. In the course of a crystallographic study on the multidrug ATP-binding cassette transporter BmrA, mass spectral analyses on samples purified with six selected detergents revealed unexpected protein contamination visible for the most part on overloaded SDS-PAGE. A major contamination from the outer membrane protein OmpF was detected in purifications with Foscholine 12 (FC12) but not with Lauryldimethylamine-N-oxide (LDAO) or any of the maltose-based detergents. Consequently, in the FC12 purified BmrA, OmpF easily crystallized over BmrA in a new space group, and whose structure is reported here. We therefore devised an optimized protocol to eliminate OmpF during the FC12 purification of BmrA. On the other hand, an additional band visible at ∼110 kDa was detected in all samples purified with the maltose-based detergents. It contained AcrB that crystallized over BmrA despite its trace amounts. Highly pure BmrA preparations could be obtained using either a ΔacrAB E. coli strain and n-dodecyl-β-D-maltopyranoside, or a classical E. coli strain and lauryl maltose neopentyl glycol for the overexpression and purification, respectively. Overall our results urge to incorporate a proteomics-based purity analysis into quality control checks prior to commencing crystallization assays of membrane proteins that are notoriously arduous to crystallize. Moreover, the strategies developed here to selectively eliminate obstinate contaminants should be applicable to the purification of other membrane proteins overexpressed in E. coli. PMID:25517996
Evaluation of ionic air purifiers for reducing aerosol exposure in confined indoor spaces.

PubMed

Grinshpun, S A; Mainelis, G; Trunov, M; Adhikari, A; Reponen, T; Willeke, K

2005-08-01

Numerous techniques have been developed over the years for reducing aerosol exposure in indoor air environments. Among indoor air purifiers of different types, ionic emitters have gained increasing attention and are presently used for removing dust particles, aeroallergens and airborne microorganisms from indoor air. In this study, five ionic air purifiers (two wearable and three stationary) that produce unipolar air ions were evaluated with respect to their ability to reduce aerosol exposure in confined indoor spaces. The concentration decay of respirable particles of different properties was monitored in real time inside the breathing zone of a human manikin, which was placed in a relatively small (2.6 m3) walk-in chamber during the operation of an ionic air purifier in calm air and under mixing air condition. The particle removal efficiency as a function of particle size was determined using the data collected with a size-selective optical particle counter. The removal efficiency of the more powerful of the two wearable ionic purifiers reached about 50% after 15 min and almost 100% after 1.5 h of continuous operation in the chamber under calm air conditions. In the absence of external ventilation, air mixing, especially vigorous one (900 CFM), enhanced the air cleaning effect. Similar results were obtained when the manikin was placed inside a partial enclosure that simulated an aircraft seating configuration. All three stationary ionic air purifiers tested in this study were found capable of reducing the aerosol concentration in a confined indoor space. The most powerful stationary unit demonstrated an extremely high particle removal efficiency that increased sharply to almost 90% within 5-6 min, reaching about 100% within 10-12 min for all particle sizes (0.3-3 microm) tested in the chamber. For the units of the same emission rate, the data suggest that the ion polarity per se (negative vs. positive) does not affect the performance but the ion emission rate does. The effects of particle size (within the tested range) and properties (NaCl, PSL, Pseudomonas fluorescens bacteria) as well as the effects of the manikin's body temperature and its breathing on the ionic purifier performance were either small or insignificant. The data suggest that the unipolar ionic air purifiers are particularly efficient in reducing aerosol exposure in the breathing zone when used inside confined spaces with a relatively high surface-to-volume ratio. Ionic air purifiers have become increasingly popular for removing dust particles, aeroallergens and airborne microorganisms from indoor air in various settings. While the indoor air cleaning effect, resulting from unipolar and bipolar ion emission, has been tested by several investigators, there are still controversial claims (favorable and unfavorable) about the performance of commercially available ionic air purifiers. Among the five tested ionic air purifiers (two wearable and three stationary) producing unipolar air ions, the units with a higher ion emission rate provided higher particle removal efficiency. The ion polarity (negative vs. positive), the particle size (0.3-3 microm) and properties (NaCl, PSL, Pseudomonas fluorescens bacteria), as well as the body temperature and breathing did not considerable affected the ionization-driven particle removal. The data suggest that the unipolar ionic air purifiers are particularly efficient in reducing aerosol exposure in the breathing zone when they are used inside confined spaces with a relatively high surface-to-volume ratio (such as automobile cabins, aircraft seating areas, bathrooms, cellular offices, small residential rooms, and animal confinements). Based on our experiments, we proposed that purifiers with a very high ion emission rate be operated in an intermittent mode if used indoors for extended time periods. As the particles migrate to and deposit on indoor surfaces during the operation of ionic air purifiers, some excessive surface contamination may occur, which introduces the need of periodic cleaning these surfaces.
Inferring Selective Constraint from Population Genomic Data Suggests Recent Regulatory Turnover in the Human Brain

PubMed Central

Schrider, Daniel R.; Kern, Andrew D.

2015-01-01

The comparative genomics revolution of the past decade has enabled the discovery of functional elements in the human genome via sequence comparison. While that is so, an important class of elements, those specific to humans, is entirely missed by searching for sequence conservation across species. Here we present an analysis based on variation data among human genomes that utilizes a supervised machine learning approach for the identification of human-specific purifying selection in the genome. Using only allele frequency information from the complete low-coverage 1000 Genomes Project data set in conjunction with a support vector machine trained from known functional and nonfunctional portions of the genome, we are able to accurately identify portions of the genome constrained by purifying selection. Our method identifies previously known human-specific gains or losses of function and uncovers many novel candidates. Candidate targets for gain and loss of function along the human lineage include numerous putative regulatory regions of genes essential for normal development of the central nervous system, including a significant enrichment of gain of function events near neurotransmitter receptor genes. These results are consistent with regulatory turnover being a key mechanism in the evolution of human-specific characteristics of brain development. Finally, we show that the majority of the genome is unconstrained by natural selection currently, in agreement with what has been estimated from phylogenetic methods but in sharp contrast to estimates based on transcriptomics or other high-throughput functional methods. PMID:26590212
Electrochemical Glucose Biosensor Based on Glucose Oxidase Displayed on Yeast Surface.

PubMed

Wang, Hongwei; Lang, Qiaolin; Liang, Bo; Liu, Aihua

2015-01-01

The conventional enzyme-based biosensor requires chemical or physical immobilization of purified enzymes on electrode surface, which often results in loss of enzyme activity and/or fractions immobilized over time. It is also costly. A major advantage of yeast surface display is that it enables the direct utilization of whole cell catalysts with eukaryote-produced proteins being displayed on the cell surface, providing an economic alternative to traditional production of purified enzymes. Herein, we describe the details of the display of glucose oxidase (GOx) on yeast cell surface and its application in the development of electrochemical glucose sensor. In order to achieve a direct electrochemistry of GOx, the entire cell catalyst (yeast-GOx) was immobilized together with multiwalled carbon nanotubes on the electrode, which allowed sensitive and selective glucose detection.
Purification and properties of aryl acylamidase from Pseudomonas fluorescens ATCC 39004.

PubMed

Hammond, P M; Price, C P; Scawen, M D

1983-05-16

Aryl acylamidase has been purified from a strain of Pseudomonas fluorescens ATCC 39004, selected from soil on the basis of its ability to utilise acylanilide compounds as a sole source of carbon. The enzyme was purified to homogeneity by a combination of ion-exchange, hydrophobic and gel-permeation chromatography. A relative molecular mass of about 52 500 was estimated by gel filtration. The native enzyme was shown to be a monomeric protein by sodium dodecyl sulphate/polyacrylamide gel electrophoresis. The enzyme was maximally active at a pH of 8.6 and at a temperature of 45 degrees C. The enzyme shows Michaelis-Menten kinetics; Km values for nitroacetanilide (69 microM) and hydroxyacetanilide (6.1 microM) were low, indicating that the enzyme has a very high affinity for both substrates.
Selection and Reduced Population Size Cannot Explain Higher Amounts of Neandertal Ancestry in East Asian than in European Human Populations

PubMed Central

Kim, Bernard Y.; Lohmueller, Kirk E.

2015-01-01

It has been hypothesized that the greater proportion of Neandertal ancestry in East Asians than in Europeans is due to the fact that purifying selection is less effective at removing weakly deleterious Neandertal alleles from East Asian populations. Using simulations of a broad range of models of selection and demography, we have shown that this hypothesis cannot account for the higher proportion of Neandertal ancestry in East Asians than in Europeans. Instead, more complex demographic scenarios, most likely involving multiple pulses of Neandertal admixture, are required to explain the data. PMID:25683122
Increased burden of deleterious variants in essential genes in autism spectrum disorder.

PubMed

Ji, Xiao; Kember, Rachel L; Brown, Christopher D; Bućan, Maja

2016-12-27

Autism spectrum disorder (ASD) is a heterogeneous, highly heritable neurodevelopmental syndrome characterized by impaired social interaction, communication, and repetitive behavior. It is estimated that hundreds of genes contribute to ASD. We asked if genes with a strong effect on survival and fitness contribute to ASD risk. Human orthologs of genes with an essential role in pre- and postnatal development in the mouse [essential genes (EGs)] are enriched for disease genes and under strong purifying selection relative to human orthologs of mouse genes with a known nonlethal phenotype [nonessential genes (NEGs)]. This intolerance to deleterious mutations, commonly observed haploinsufficiency, and the importance of EGs in development suggest a possible cumulative effect of deleterious variants in EGs on complex neurodevelopmental disorders. With a comprehensive catalog of 3,915 mammalian EGs, we provide compelling evidence for a stronger contribution of EGs to ASD risk compared with NEGs. By examining the exonic de novo and inherited variants from 1,781 ASD quartet families, we show a significantly higher burden of damaging mutations in EGs in ASD probands compared with their non-ASD siblings. The analysis of EGs in the developing brain identified clusters of coexpressed EGs implicated in ASD. Finally, we suggest a high-priority list of 29 EGs with potential ASD risk as targets for future functional and behavioral studies. Overall, we show that large-scale studies of gene function in model organisms provide a powerful approach for prioritization of genes and pathogenic variants identified by sequencing studies of human disease.
Increased burden of deleterious variants in essential genes in autism spectrum disorder

PubMed Central

Kember, Rachel L.; Brown, Christopher D.; Bućan, Maja

2016-01-01

Autism spectrum disorder (ASD) is a heterogeneous, highly heritable neurodevelopmental syndrome characterized by impaired social interaction, communication, and repetitive behavior. It is estimated that hundreds of genes contribute to ASD. We asked if genes with a strong effect on survival and fitness contribute to ASD risk. Human orthologs of genes with an essential role in pre- and postnatal development in the mouse [essential genes (EGs)] are enriched for disease genes and under strong purifying selection relative to human orthologs of mouse genes with a known nonlethal phenotype [nonessential genes (NEGs)]. This intolerance to deleterious mutations, commonly observed haploinsufficiency, and the importance of EGs in development suggest a possible cumulative effect of deleterious variants in EGs on complex neurodevelopmental disorders. With a comprehensive catalog of 3,915 mammalian EGs, we provide compelling evidence for a stronger contribution of EGs to ASD risk compared with NEGs. By examining the exonic de novo and inherited variants from 1,781 ASD quartet families, we show a significantly higher burden of damaging mutations in EGs in ASD probands compared with their non-ASD siblings. The analysis of EGs in the developing brain identified clusters of coexpressed EGs implicated in ASD. Finally, we suggest a high-priority list of 29 EGs with potential ASD risk as targets for future functional and behavioral studies. Overall, we show that large-scale studies of gene function in model organisms provide a powerful approach for prioritization of genes and pathogenic variants identified by sequencing studies of human disease. PMID:27956632
Differential Activities of the Two Closely Related Withanolides, Withaferin A and Withanone: Bioinformatics and Experimental Evidences

PubMed Central

Manjunath, Kavyashree; Uthayakumar, M.; Kanaujia, Shankar P.; Kaul, Sunil C.; Sekar, Kanagaraj; Wadhwa, Renu

2012-01-01

Background and Purpose Withanolides are naturally occurring chemical compounds. They are secondary metabolites produced via oxidation of steroids and structurally consist of a steroid-backbone bound to a lactone or its derivatives. They are known to protect plants against herbivores and have medicinal value including anti-inflammation, anti-cancer, adaptogenic and anti-oxidant effects. Withaferin A (Wi-A) and Withanone (Wi-N) are two structurally similar withanolides isolated from Withania somnifera, also known as Ashwagandha in Indian Ayurvedic medicine. Ashwagandha alcoholic leaf extract (i-Extract), rich in Wi-N, was shown to kill cancer cells selectively. Furthermore, the two closely related purified phytochemicals, Wi-A and Wi-N, showed differential activity in normal and cancer human cells in vitro and in vivo. We had earlier identified several genes involved in cytotoxicity of i-Extract in human cancer cells by loss-of-function assays using either siRNA or randomized ribozyme library. Methodology/Principal Findings In the present study, we have employed bioinformatics tools on four genes, i.e., mortalin, p53, p21 and Nrf2, identified by loss-of-function screenings. We examined the docking efficacy of Wi-N and Wi-A to each of the four targets and found that the two closely related phytochemicals have differential binding properties to the selected cellular targets that can potentially instigate differential molecular effects. We validated these findings by undertaking parallel experiments on specific gene responses to either Wi-N or Wi-A in human normal and cancer cells. We demonstrate that Wi-A that binds strongly to the selected targets acts as a strong cytotoxic agent both for normal and cancer cells. Wi-N, on the other hand, has a weak binding to the targets; it showed milder cytotoxicity towards cancer cells and was safe for normal cells. The present molecular docking analyses and experimental evidence revealed important insights to the use of Wi-A and Wi-N for cancer treatment and development of new anti-cancer phytochemical cocktails. PMID:22973447
Population-genomic variation within RNA viruses of the Western honey bee, Apis mellifera, inferred from deep sequencing

PubMed Central

2013-01-01

Background Deep sequencing of viruses isolated from infected hosts is an efficient way to measure population-genetic variation and can reveal patterns of dispersal and natural selection. In this study, we mined existing Illumina sequence reads to investigate single-nucleotide polymorphisms (SNPs) within two RNA viruses of the Western honey bee (Apis mellifera), deformed wing virus (DWV) and Israel acute paralysis virus (IAPV). All viral RNA was extracted from North American samples of honey bees or, in one case, the ectoparasitic mite Varroa destructor. Results Coverage depth was generally lower for IAPV than DWV, and marked gaps in coverage occurred in several narrow regions (< 50 bp) of IAPV. These coverage gaps occurred across sequencing runs and were virtually unchanged when reads were re-mapped with greater permissiveness (up to 8% divergence), suggesting a recurrent sequencing artifact rather than strain divergence. Consensus sequences of DWV for each sample showed little phylogenetic divergence, low nucleotide diversity, and strongly negative values of Fu and Li’s D statistic, suggesting a recent population bottleneck and/or purifying selection. The Kakugo strain of DWV fell outside of all other DWV sequences at 100% bootstrap support. IAPV consensus sequences supported the existence of multiple clades as had been previously reported, and Fu and Li’s D was closer to neutral expectation overall, although a sliding-window analysis identified a significantly positive D within the protease region, suggesting selection maintains diversity in that region. Within-sample mean diversity was comparable between the two viruses on average, although for both viruses there was substantial variation among samples in mean diversity at third codon positions and in the number of high-diversity sites. FST values were bimodal for DWV, likely reflecting neutral divergence in two low-diversity populations, whereas IAPV had several sites that were strong outliers with very low FST. Conclusions This initial survey of genetic variation within honey bee RNA viruses suggests future directions for studies examining the underlying causes of population-genetic structure in these economically important pathogens. PMID:23497218
Population-genomic variation within RNA viruses of the Western honey bee, Apis mellifera, inferred from deep sequencing.

PubMed

Cornman, Robert Scott; Boncristiani, Humberto; Dainat, Benjamin; Chen, Yanping; vanEngelsdorp, Dennis; Weaver, Daniel; Evans, Jay D

2013-03-07

Deep sequencing of viruses isolated from infected hosts is an efficient way to measure population-genetic variation and can reveal patterns of dispersal and natural selection. In this study, we mined existing Illumina sequence reads to investigate single-nucleotide polymorphisms (SNPs) within two RNA viruses of the Western honey bee (Apis mellifera), deformed wing virus (DWV) and Israel acute paralysis virus (IAPV). All viral RNA was extracted from North American samples of honey bees or, in one case, the ectoparasitic mite Varroa destructor. Coverage depth was generally lower for IAPV than DWV, and marked gaps in coverage occurred in several narrow regions (< 50 bp) of IAPV. These coverage gaps occurred across sequencing runs and were virtually unchanged when reads were re-mapped with greater permissiveness (up to 8% divergence), suggesting a recurrent sequencing artifact rather than strain divergence. Consensus sequences of DWV for each sample showed little phylogenetic divergence, low nucleotide diversity, and strongly negative values of Fu and Li's D statistic, suggesting a recent population bottleneck and/or purifying selection. The Kakugo strain of DWV fell outside of all other DWV sequences at 100% bootstrap support. IAPV consensus sequences supported the existence of multiple clades as had been previously reported, and Fu and Li's D was closer to neutral expectation overall, although a sliding-window analysis identified a significantly positive D within the protease region, suggesting selection maintains diversity in that region. Within-sample mean diversity was comparable between the two viruses on average, although for both viruses there was substantial variation among samples in mean diversity at third codon positions and in the number of high-diversity sites. FST values were bimodal for DWV, likely reflecting neutral divergence in two low-diversity populations, whereas IAPV had several sites that were strong outliers with very low FST. This initial survey of genetic variation within honey bee RNA viruses suggests future directions for studies examining the underlying causes of population-genetic structure in these economically important pathogens.
Aptamer facilitated purification of functional proteins.

PubMed

Beloborodov, Stanislav S; Bao, Jiayin; Krylova, Svetlana M; Shala-Lawrence, Agnesa; Johnson, Philip E; Krylov, Sergey N

2018-01-15

DNA aptamers are attractive capture probes for affinity chromatography since, in contrast to antibodies, they can be chemically synthesized and, in contrast to tag-specific capture probes (such as Nickel-NTA or Glutathione), they can be used for purification of proteins free of genetic modifications (such as His or GST tags). Despite these attractive features of aptamers as capture probes, there are only a few reports on aptamer-based protein purification and none of them includes a test of the purified protein's activity, thus, leaving discouraging doubts about method's ability to purify proteins in their active state. The goal of this work was to prove that aptamers could facilitate isolation of active proteins. We refined a complete aptamer-based affinity purification procedure, which takes 4 h to complete. We further applied this procedure to purify two recombinant proteins, MutS and AlkB, from bacterial cell culture: 0.21 mg of 85%-pure AlkB from 4 mL of culture and 0.24 mg of 82%-pure MutS from 0.5 mL of culture. Finally, we proved protein activity by two capillary electrophoresis based assays: an enzymatic assay for AlkB and a DNA-binding assay for MutS. We suggest that in combination with aptamer selection for non-purified protein targets in crude cell lysate, aptamer-based purification provides a means of fast isolation of tag-free recombinant proteins in their native state without the use of antibodies. Copyright © 2017 Elsevier B.V. All rights reserved.
Expression, Identification and Purification of Dictyostelium Acetoacetyl-CoA Thiolase Expressed in Escherichia coli

PubMed Central

Tanaka, Takeshi; Shima, Yasuyuki; Ogawa, Naoki; Nagayama, Koki; Yoshida, Takashi; Ohmachi, Tetsuo

2011-01-01

Acetoacetyl-CoA thiolase (AT) is an enzyme that catalyses the CoA-dependent thiolytic cleavage of acetoacetyl-CoA to yield 2 molecules of acetyl-CoA, or the reverse condensation reaction. A full-length cDNA clone pBSGT-3, which has homology to known thiolases, was isolated from Dictyostelium cDNA library. Expression of the protein encoded in pBSGT-3 in Escherichia coli, its thiolase enzyme activity, and the amino acid sequence homology search revealed that pBSGT-3 encodes an AT. The recombinant AT (r-thiolase) was expressed in an active form in an E. coli expression system, and purified to homogeneity by selective ammonium sulfate fractionation and two steps of column chromatography. The purified enzyme exhibited a specific activity of 4.70 mU/mg protein. Its N-terminal sequence was (NH2)-Arg-Met-Tyr-Thr-Thr-Ala-Lys-Asn-Leu-Glu-, which corresponds to the sequence from positions 15 to 24 of the amino acid sequence deduced from pBSGT-3 clone. The r-thiolase in the inclusion body expressed highly in E. coli was the precursor form, which is slightly larger than the purified r-thiolase. When incubated with the cell-free extract of Dictyostelium cells, the precursor was converted to the same size to the purified r-thiolase, suggesting that the presequence at the N-terminus is removed by a Dictyostelium processing peptidase. PMID:21209787
Application of aqueous biphasic systems as strategy to purify tannase from Aspergillus tamarii URM 7115.

PubMed

de Sena, Amanda Reges; Barros Oliveira, Flávio Manoel; Campos Leite, Tonny Cley; Evaristo da Silva Nascimento, Talita Camila; Moreira, Keila Aparecida; de Assis, Sandra Aparecida

2017-10-21

The aims of the current study are to assess the influence of polyethylene glycol (PEG) concentration, molar mass, pH, and citrate concentrations on aqueous biphasic systems based on 2 4 factorial designs, as well as to check their capacity to purify tannase secreted by Aspergillus tamarii URM 7115. Tannase was produced through submerged fermentation at 26°C for 67 h in Czapeck-Dox modified broth and added with yeast extract and tannic acid. The factorial design was followed to assess the influence of PEG molar mass (M PEG 600; 4,000 and 8,000 g/ mol), and PEG (C PEG 20.0; 22.0 and 24.0% w/w) and citrate concentrations (C CIT 15.0, 17.5, and 20.0%, w/w), as well as of pH (6.0, 7.0, and 8.0) on the response variables; moreover, partition coefficient (K), yield (Y), and purification factor (PF) were analyzed. The most suitable parameters to purify tannase secreted by A. tamarii URM 7115 through a biphasic system were 600 (g/mol) M PEG , 24% (w/w) C PEG , 15% (w/w) C CIT at pH 6.0 and they resulted in 6.33 enzyme partition, 131.25% yield, 19.80 purification factor and 195.08 selectivity. Tannase secreted by A. tamarii URM 7115 purified through aqueous biphasic systems composed of PEG/citrate can be used for industrial purposes, since it presents suitable purification factor and yield.
A Ten-Week Biochemistry Lab Project Studying Wild-Type and Mutant Bacterial Alkaline Phosphatase

ERIC Educational Resources Information Center

Witherow, D. Scott

2016-01-01

This work describes a 10-week laboratory project studying wild-type and mutant bacterial alkaline phosphatase, in which students purify, quantitate, and perform kinetic assays on wild-type and selected mutants of the enzyme. Students also perform plasmid DNA purification, digestion, and gel analysis. In addition to simply learning important…
Method for removal of phosgene from boron trichloride. [DOE patent application; mercury arc lamp

DOEpatents

Freund, S.M.

1981-09-03

Selective ultraviolet photolysis using an unfiltered mercury arc lamp has been used to substantially reduce the phosgene impurity in a mixture of boron trichloride and phosgene. Infrared spectrophotometric analysis of the sample before and after irradiation shows that it is possible to highly purify commercially available boron trichloride with this method.
Molecular and biological characterization of 'Rs551, a filamentous bacteriophage isolated from a race 3 biovar 2 strain of Ralstonia solanacearum

USDA-ARS?s Scientific Manuscript database

A filamentous bacteriophage, designated 'Rs551, was isolated and purified from the quarantine and select agent phytopathogen Ralstonia solanacearum race 3 biovar 2 strain UW551 (phylotype IIB sequevar 1) grown under normal culture conditions. Electron microscopy suggested that 'Rs551 is a member of ...
Purification and Kinetic Properties of Serine Acetyltransferase Free of O-Acetylserine(thiol)lyase from Spinach Chloroplasts.

PubMed

Ruffet, M. L.; Droux, M.; Douce, R.

1994-02-01

Serine acetyltransferase, a key enzyme in the L-cysteine biosynthetic pathway, was purified over 300,000-fold from the stroma of spinach (Spinacia oleracea) leaf chloroplasts. The purification procedure consisted of ammonium sulfate precipitation, anion-exchange chromatography (Trisacryl M DEAE and Mono Q HR10/10), hydroxylapatite chromatography, and gel filtration (Superdex 200). The purified enzyme exhibited a specific activity higher than 200 units mg-1 and a subunit molecular mass of about 33 kD upon polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Moreover, the purified serine acetyltransferase appeared to be essentially free of O-acetyleserine(thiol)lyase, another enzyme component in the L-cysteine biosynthetic pathway. A steady-state kinetic analysis indicated that the mechanism of the enzyme-catalyzed reaction involves a double displacement. The apparent Km for the two substrates, L-serine and acetyl-coenzyme A, were 2.29 [plus or minus] 0.43 and 0.35 [plus or minus] 0.02 mM, respectively. The rate of L-cysteine synthesis in vitro was measured in a coupled enzyme assay using extensively purified O-acetylserine(thiol)lyase and serine acetyltransferase. This rate was maximum when the assay contained approximately a 400-fold excess of O-acetylserine(thiol)lyase over serine acetyltransferase. Measurements of the relative level of O-acetylserine(thiol)lyase and serine acetyltransferase activities in the stroma indicated that the former enzyme was present in much larger quantities than the latter. Thus, the activity ratio for these two enzymes [O-acetylserine(thiol)lyase activity/serine acetyltransferase activity] measured in the stromal protein extract was 345. This strongly suggested that all the O-acetylserine(thiol)lyase and serine acetyltransferase activities in the stroma are involved in bringing a full synthesis of L-cysteine in the chloroplast.
Purification and Kinetic Properties of Serine Acetyltransferase Free of O-Acetylserine(thiol)lyase from Spinach Chloroplasts.

PubMed Central

Ruffet, M. L.; Droux, M.; Douce, R.

1994-01-01

Serine acetyltransferase, a key enzyme in the L-cysteine biosynthetic pathway, was purified over 300,000-fold from the stroma of spinach (Spinacia oleracea) leaf chloroplasts. The purification procedure consisted of ammonium sulfate precipitation, anion-exchange chromatography (Trisacryl M DEAE and Mono Q HR10/10), hydroxylapatite chromatography, and gel filtration (Superdex 200). The purified enzyme exhibited a specific activity higher than 200 units mg-1 and a subunit molecular mass of about 33 kD upon polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Moreover, the purified serine acetyltransferase appeared to be essentially free of O-acetyleserine(thiol)lyase, another enzyme component in the L-cysteine biosynthetic pathway. A steady-state kinetic analysis indicated that the mechanism of the enzyme-catalyzed reaction involves a double displacement. The apparent Km for the two substrates, L-serine and acetyl-coenzyme A, were 2.29 [plus or minus] 0.43 and 0.35 [plus or minus] 0.02 mM, respectively. The rate of L-cysteine synthesis in vitro was measured in a coupled enzyme assay using extensively purified O-acetylserine(thiol)lyase and serine acetyltransferase. This rate was maximum when the assay contained approximately a 400-fold excess of O-acetylserine(thiol)lyase over serine acetyltransferase. Measurements of the relative level of O-acetylserine(thiol)lyase and serine acetyltransferase activities in the stroma indicated that the former enzyme was present in much larger quantities than the latter. Thus, the activity ratio for these two enzymes [O-acetylserine(thiol)lyase activity/serine acetyltransferase activity] measured in the stromal protein extract was 345. This strongly suggested that all the O-acetylserine(thiol)lyase and serine acetyltransferase activities in the stroma are involved in bringing a full synthesis of L-cysteine in the chloroplast. PMID:12232109
Cannabidivarin-rich cannabis extracts are anticonvulsant in mouse and rat via a CB1 receptor-independent mechanism

PubMed Central

Hill, T D M; Cascio, M-G; Romano, B; Duncan, M; Pertwee, R G; Williams, C M; Whalley, B J; Hill, A J

2013-01-01

BACKGROUND AND PURPOSE Epilepsy is the most prevalent neurological disease and is characterized by recurrent seizures. Here, we investigate (i) the anticonvulsant profiles of cannabis-derived botanical drug substances (BDSs) rich in cannabidivarin (CBDV) and containing cannabidiol (CBD) in acute in vivo seizure models and (ii) the binding of CBDV BDSs and their components at cannabinoid CB1 receptors. EXPERIMENTAL APPROACH The anticonvulsant profiles of two CBDV BDSs (50–422 mg·kg−1) were evaluated in three animal models of acute seizure. Purified CBDV and CBD were also evaluated in an isobolographic study to evaluate potential pharmacological interactions. CBDV BDS effects on motor function were also investigated using static beam and grip strength assays. Binding of CBDV BDSs to cannabinoid CB1 receptors was evaluated using displacement binding assays. KEY RESULTS CBDV BDSs exerted significant anticonvulsant effects in the pentylenetetrazole (≥100 mg·kg−1) and audiogenic seizure models (≥87 mg·kg−1), and suppressed pilocarpine-induced convulsions (≥100 mg·kg−1). The isobolographic study revealed that the anticonvulsant effects of purified CBDV and CBD were linearly additive when co-administered. Some motor effects of CBDV BDSs were observed on static beam performance; no effects on grip strength were found. The Δ9-tetrahydrocannabinol and Δ9-tetrahydrocannabivarin content of CBDV BDS accounted for its greater affinity for CB1 cannabinoid receptors than purified CBDV. CONCLUSIONS AND IMPLICATIONS CBDV BDSs exerted significant anticonvulsant effects in three models of seizure that were not mediated by the CB1 cannabinoid receptor and were of comparable efficacy with purified CBDV. These findings strongly support the further clinical development of CBDV BDSs for the treatment of epilepsy. PMID:23902406

Characterization of an extracellular lipase from the biocontrol fungus, Nomuraea rileyi MJ, and its toxicity toward Spodoptera litura.

PubMed

Supakdamrongkul, Piyaporn; Bhumiratana, Amaret; Wiwat, Chanpen

2010-11-01

An extracellular lipase from Nomuraea rileyi MJ was purified 23.9-fold with 1.69% yield by ammonium sulfate precipitation followed by Sephacryl S-100 HR column chromatography. By mass spectrometry and SDS-polyacrylamide gel electrophoresis, the molecular weight of the homogenous lipase was 81kDa. The N-terminal sequence was determined as LeuSerValGluGlnThrLysLeuSerLysLeuAlaTyrAsnAsp and it showed no homology to sequences of known lipases. The optimum pH and temperature for activity were 8.0 and 35°C, respectively. The enzyme was stable in the pH range 7.0-9.0 and at 15-35°C for 1h. Higher activity was observed in the presence of surfactants, Na(+), NH(4)(+) ions, NaN(3) and ethylenediaminetetraacetic acid (EDTA), while Co(2+) and Cu(2+) ions, cysteine and dithiothreitol (DTT) strongly inhibited activity. The purified lipase hydrolyzed both synthetic and natural triglycerides with maximum activity for trilaurin and coconut oil, respectively. It also hydrolyzed esters of p-nitrophenol (pNP) with highest activity for p-nitrophenyl caprate (pNPCA). The purified lipase was found to promote N. rileyi spore germination in vitro in that germination reached 98% in conidial suspensions containing purified lipase at 2.75 U. Moreover, it enhanced toxicity of N. rileyi toward Spodoptera litura larvae with mortality via topical application reaching 63.3% at 4-10days post-treatment which calculated to be 2.7 times higher than the mortality obtained using conidial suspensions alone. Copyright © 2010 Elsevier Inc. All rights reserved.
Antinociceptive and anti-inflammatory effect of sulfated polysaccharide fractions from Sargassum wightii and Halophila ovalis in male Wistar rats

PubMed Central

Neelakandan, Yuvaraj; Venkatesan, Arul

2016-01-01

Aim: The aim of this objective is to evaluate the antinociceptive and anti-inflammatory potential of sulfated polysaccharide purified fractions isolated from brown seaweed Sargassum wightii and seagrass Halophila ovalis in male Wistar rats. Subjects and Methods: Crude sulfated polysaccharide from S. wightii and H. ovalis was subjected to anion exchange chromatography, and the chemical composition was investigated. The antinociceptive activity of purified fractions was investigated using formalin and hot plate test. Carrageenan-induced paw edema, peritonitis model, and Freund's Complete Adjuvant-induced arthritis model were employed to determine the anti-inflammatory activity. Results: In the formalin test, there was a significant reduction in licking time in both phases of the test at a dose of 10 mg/kg. In the hot plate test, the antinociceptive effect was observed only in animals treated with 5, 10 mg/kg suggesting that the analgesic effect occurs through a central action mechanism. Sw FrIV and Ho FrIV significantly inhibited paw edema induced by carrageenan, especially at 3 h after treatment and potentially decreased neutrophil migration at 10 mg/kg, respectively. In Freund's adjuvant-induced arthritic rats, a significant reduction in paw volume was observed in Sw FrIV and Ho FrIV-treated groups (10 mg/kg). Conclusion: Purified components from S. wightii and H. ovalis have strong antinociceptive and anti-inflammatory effect on animal model. However, to determine the molecular mechanism, it is necessary to investigate the effect of purified fractions on inhibition of nitric oxide synthase expression mediated by inhibiting the phosphorylation of various signal proteins in lipopolysaccharide-stimulated RAW264.7 cells. PMID:27721544
Purification and characterization of an amidohydrolase for N4-long-chain fatty acyl derivatives of 1-beta-D-arabinofuranosylcytosine from mouse liver microsomes.

PubMed

Hori, K; Tsuruo, T; Tsukagoshi, S; Sakurai, Y

1984-03-01

N4-Long-chain fatty acyl-1-beta-D-arabinofuranosylcytosine amidohydrolase, a metabolizing enzyme for N4-acyl derivatives of 1-beta-D-arabinofuranosylcytosine with long-chain fatty acids, was purified from mouse liver microsomes. The purification was accomplished by solubilization of liver microsomes with Triton X-100, diethylaminoethyl cellulose chromatography, gel filtrations, hydroxyapatite chromatography, and concanavalin A:Sepharose chromatography. On sodium dodecyl sulfate:polyacrylamide gel electrophoresis, the purified enzyme preparation produced a single protein band with a molecular weight of 54,000. The enzyme had an optimal pH of 9.0, and the Michaelis constant for N4-palmitoyl-1-beta-D-arabinofuranosylcytosine was 67 microM. The thiols such as dithiothreitol or 2-mercaptoethanol stabilized the enzyme and stimulated its activity. p-Chloromercuribenzoate, N-ethylmaleimide, diisopropylfluorophosphate, and phenylmethylsulfonyl fluoride strongly inhibited the reaction. Bovine serum albumin markedly stimulated the enzyme activity, whereas detergents such as Triton X-100, deoxycholate, and sodium dodecyl sulfate had little effect. The enzyme did not require monovalent or divalent cations. Among the series of N4-acyl derivatives of 1-beta-D-arabinofuranosylcytosine with different chain lengths of acyl residues, the purified enzyme preferentially hydrolyzed the derivatives with long-chain fatty acids (C12 to C18), and N4-palmitoyl-1-beta-D-arabinofuranosylcytosine was the most susceptible. The purified enzyme was inactive on various N-acylamino acids, amides, oligopeptides, proteins, N-acylsphingosines (ceramides), triglyceride, lecithin, and lysolecithin. These results suggest that N4-long-chain fatty acyl-1-beta-D-arabinofuranosylcytosine amidohydrolase may be a new type of linear amidase.
Parallel evolution or purifying selection, not introgression, explain similarity in the pyrethroid detoxification linked GSTE4 of Anopheles gambiae and An. arabiensis

PubMed Central

Wilding, C.S.; Weetman, D.; Rippon, E.J.; Steen, K.; Mawejje, H.D.; Barsukov, I.; Donnelly, M.J.

2014-01-01

Insecticide resistance is a major impediment to the control of vectors and pests of public health importance and is a strongly selected trait capable of rapid spread, sometimes even between closely-related species. Elucidating the mechanisms generating insecticide resistance in mosquito vectors of disease, and understanding the spread of resistance within and between populations and species are vital for the development of robust resistance management strategies. Here we studied the mechanisms of resistance in two sympatric members of the Anopheles gambiae species complex – the major vector of malaria in sub-Saharan Africa – in order to understand how resistance has developed and spread in eastern Uganda, a region with some of the highest levels of malaria. In eastern Uganda, where the mosquitoes Anopheles arabiensis and An. gambiae can be found sympatrically, low levels of hybrids (0.4%) occur, offering a route for introgression of adaptively important variants between species. In independent microarray studies of insecticide resistance, Gste4, an insect-specific glutathione S-transferase, was among the most significantly up-regulated genes in both species. To test the hypothesis of interspecific introgression, we sequenced 2.3kbp encompassing Gste4. Whilst this detailed sequencing ruled out introgression, we detected strong positive selection acting on Gste4. However, these sequences, followed by haplotype-specific qPCR, showed that the apparent up-regulation in An. arabiensis is a result of allelic variation across the microarray probe binding sites which artefactually elevates the gene expression signal. Thus, face-value acceptance of microarray data can be misleading and it is advisable to conduct a more detailed investigation of the causes and nature of such signal. The identification of positive selection acting on this locus led us to functionally express and characterise allelic variants of GSTE4. Although the in vitro data do not support a direct role for GSTE4 in metabolism, they do support a role for this enzyme in insecticide sequestration. Thus, the demonstration of a role for an up-regulated gene in metabolic resistance to insecticides should not be limited to simply whether it can metabolise insecticide; such a strict criterion would argue against the involvement of GSTE4 despite the weight of evidence to the contrary. PMID:25213601
α-l-rhamnosidase selective for rutin to isoquercitrin transformation from Penicillium griseoroseum MTCC-9224.

PubMed

Yadav, Sarita; Yadava, Sudha; Yadav, Kapil D S

2017-02-01

An α-l-rhamnosidase secreting fungal strain has been isolated from the decaying goose berry (Emblica officinalis) fruit peel. The fungal strain has been identified as Penicillium greoroseum MTCC-9224. The α-l-rhamnosidase of this fungal strain has been purified to homogeneity using a simple procedure involving concentration by ultra filtration and an anion exchange chromatography on DEAE-cellulose. The purified enzyme gave a single protein band corresponding to molecular mass of 97kDa in SDS-PAGE analysis. The native-PAGE analysis also gave a single protein band confirming the purity of the enzyme. Using p-nitrophenyl-α-l-rhamnopyranoside as the substrate, K m and k cat values of the enzyme were 0.65mM and 43.65s -1 , respectively. The pH and temperature optima of the enzyme were 6.5 and 57°C, respectively. The activation energy for the thermal denaturation of the enzyme was 27.9kJ/mol. The purified α-l-rhamnosidase hydrolyzed rutin to isoquercitrin and l-rhamnose but has no effect on naringin and hesperidin. Copyright © 2017 Elsevier Inc. All rights reserved.
Derivation of highly purified cardiomyocytes from human induced pluripotent stem cells using small molecule-modulated differentiation and subsequent glucose starvation.

PubMed

Sharma, Arun; Li, Guang; Rajarajan, Kuppusamy; Hamaguchi, Ryoko; Burridge, Paul W; Wu, Sean M

2015-03-18

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have become an important cell source to address the lack of primary cardiomyocytes available for basic research and translational applications. To differentiate hiPSCs into cardiomyocytes, various protocols including embryoid body (EB)-based differentiation and growth factor induction have been developed. However, these protocols are inefficient and highly variable in their ability to generate purified cardiomyocytes. Recently, a small molecule-based protocol utilizing modulation of Wnt/β-Catenin signaling was shown to promote cardiac differentiation with high efficiency. With this protocol, greater than 50%-60% of differentiated cells were cardiac troponin-positive cardiomyocytes were consistently observed. To further increase cardiomyocyte purity, the differentiated cells were subjected to glucose starvation to specifically eliminate non-cardiomyocytes based on the metabolic differences between cardiomyocytes and non-cardiomyocytes. Using this selection strategy, we consistently obtained a greater than 30% increase in the ratio of cardiomyocytes to non-cardiomyocytes in a population of differentiated cells. These highly purified cardiomyocytes should enhance the reliability of results from human iPSC-based in vitro disease modeling studies and drug screening assays.
Metabolism of aspartame by human and pig intestinal microvillar peptidases.

PubMed Central

Hooper, N M; Hesp, R J; Tieku, S

1994-01-01

The artificial sweetener aspartame (N-L-alpha-aspartyl-L-phenyl-alanine-1-methyl ester; Nutrasweet), its decomposition product alpha Asp-Phe and the related peptide alpha Asp-PheNH2 were rapidly hydrolysed by microvillar membranes prepared from human duodenum, jejunum and ileum, and from pig duodenum and kidney. The metabolism of aspartame by the human and pig intestinal microvillar membrane preparations was inhibited significantly (> 78%) by amastatin or 1,10-phenanthroline, and partially (> 38%) by actinonin or bestatin, and was activated 2.9-4.5-fold by CaCl2. The inhibition by amastatin and 1,10-phenanthroline, and the activation by CaCl2 are characteristic of the cell-surface ectoenzyme aminopeptidase A (EC 3.4.11.7) and a purified preparation of this enzyme hydrolysed aspartame with a Km of 0.25 mM and a Vmax of 126 mumol/min per mg. A purified preparation of aminopeptidase W (EC 3.4.11.16) also hydrolysed aspartame but with a Km of 4.96 mM and a Vmax of 110 mumol/min per mg. However, rentiapril, an inhibitor of aminopeptidase W, caused only slight inhibition (maximally 19%) of the hydrolysis of aspartame by the microvillar membrane preparations. Similar patterns of inhibition and kinetic parameters were observed for alpha Asp-Phe and alpha Asp-PheNH2. Two other decomposition products of aspartame, beta Asp-PheMe and cyclo-Asp-Phe, were essentially resistant to hydrolysis by both the human and pig intestinal microvillar membrane preparations and the purified preparations of aminopeptidases A and W. Although the relatively selective inhibitor of aminopeptidase N (EC 3.4.11.2), actinonin, partially inhibited the metabolism of aspartame, alpha Asp-Phe and alpha Asp-PheNH2 by the human and pig intestinal microvillar membrane preparations, these peptides were not hydrolysed by a purified preparation of aminopeptidase N. Membrane dipeptidase (EC 3.4.13.19) only hydrolysed the unblocked dipeptide, alpha Asp-Phe, but the selective inhibitor of this enzyme, cilastatin, did not block the metabolism of alpha Asp-Phe by the microvillar membrane preparations. PMID:8141778
Isolation and Identification of Aspergillus fumigatus Mycotoxins on Growth Medium and Some Building Materials

PubMed Central

Nieminen, Susanna M.; Kärki, Riikka; Auriola, Seppo; Toivola, Mika; Laatsch, Hartmut; Laatikainen, Reino; Hyvärinen, Anne; von Wright, Atte

2002-01-01

Genotoxic and cytotoxic compounds were isolated and purified from the culture medium of an indoor air mold, Aspergillus fumigatus. One of these compounds was identified as gliotoxin, a known fungal secondary metabolite. Growth of A. fumigatus and gliotoxin production on some building materials were also studied. Strong growth of the mold and the presence of gliotoxin were detected on spruce wood, gypsum board, and chipboard under saturation conditions. PMID:12324333
Myosin light chain 2-based selection of human iPSC-derived early ventricular cardiac myocytes

PubMed Central

Bizy, Alexandra; Guerrero-Serna, Guadalupe; Hu, Bin; Ponce-Balbuena, Daniela; Willis, B. Cicero; Zarzoso, Manuel; Ramirez, Rafael J.; Sener, Michelle F.; Mundada, Lakshmi V.; Klos, Matthew; Devaney, Eric J.; Vikstrom, Karen L.; Herron, Todd J.; Jalife, José

2014-01-01

Applications of human induced pluripotent stemcell derived-cardiac myocytes (hiPSC-CMs) would be strengthened by the ability to generate specific cardiac myocyte (CM) lineages. However, purification of lineage-specific hiPSC-CMs is limited by the lack of cell marking techniques. Here, we have developed an iPSC-CM marking system using recombinant adenoviral reporter constructs with atrial- or ventricular-specific myosin light chain-2 (MLC-2) promoters. MLC-2a and MLC-2v selected hiPSC-CMs were purified by fluorescence-activated cell sorting and their biochemical and electrophysiological phenotypes analyzed. We demonstrate that the phenotype of both populations remained stable in culture and they expressed the expected sarcomeric proteins, gap junction proteins and chamber-specific transcription factors. Compared to MLC-2a cells, MLC-2v selected CMs had larger action potential amplitudes and durations. In addition, by immunofluorescence, we showed that MLC-2 isoform expression can be used to enrich hiPSC-CM consistent with early atrial and ventricularmyocyte lineages. However, only the ventricular myosin light chain-2 promoter was able to purify a highly homogeneous population of iPSC-CMs. Using this approach, it is now possible to develop ventricular-specific disease models using iPSC-CMs while atrial-specific iPSC-CM cultures may require additional chamber-specific markers. PMID:24095945
Myosin light chain 2-based selection of human iPSC-derived early ventricular cardiac myocytes.

PubMed

Bizy, Alexandra; Guerrero-Serna, Guadalupe; Hu, Bin; Ponce-Balbuena, Daniela; Willis, B Cicero; Zarzoso, Manuel; Ramirez, Rafael J; Sener, Michelle F; Mundada, Lakshmi V; Klos, Matthew; Devaney, Eric J; Vikstrom, Karen L; Herron, Todd J; Jalife, José

2013-11-01

Applications of human induced pluripotent stem cell derived-cardiac myocytes (hiPSC-CMs) would be strengthened by the ability to generate specific cardiac myocyte (CM) lineages. However, purification of lineage-specific hiPSC-CMs is limited by the lack of cell marking techniques. Here, we have developed an iPSC-CM marking system using recombinant adenoviral reporter constructs with atrial- or ventricular-specific myosin light chain-2 (MLC-2) promoters. MLC-2a and MLC-2v selected hiPSC-CMs were purified by fluorescence-activated cell sorting and their biochemical and electrophysiological phenotypes analyzed. We demonstrate that the phenotype of both populations remained stable in culture and they expressed the expected sarcomeric proteins, gap junction proteins and chamber-specific transcription factors. Compared to MLC-2a cells, MLC-2v selected CMs had larger action potential amplitudes and durations. In addition, by immunofluorescence, we showed that MLC-2 isoform expression can be used to enrich hiPSC-CM consistent with early atrial and ventricular myocyte lineages. However, only the ventricular myosin light chain-2 promoter was able to purify a highly homogeneous population of iPSC-CMs. Using this approach, it is now possible to develop ventricular-specific disease models using iPSC-CMs while atrial-specific iPSC-CM cultures may require additional chamber-specific markers. © 2013.
Ceruloplasmin Is an Endogenous Inhibitor of Myeloperoxidase*

PubMed Central

Chapman, Anna L. P.; Mocatta, Tessa J.; Shiva, Sruti; Seidel, Antonia; Chen, Brian; Khalilova, Irada; Paumann-Page, Martina E.; Jameson, Guy N. L.; Winterbourn, Christine C.; Kettle, Anthony J.

2013-01-01

Myeloperoxidase is a neutrophil enzyme that promotes oxidative stress in numerous inflammatory pathologies. It uses hydrogen peroxide to catalyze the production of strong oxidants including chlorine bleach and free radicals. A physiological defense against the inappropriate action of this enzyme has yet to be identified. We found that myeloperoxidase oxidized 75% of the ascorbate in plasma from ceruloplasmin knock-out mice, but there was no significant loss in plasma from wild type animals. When myeloperoxidase was added to human plasma it became bound to other proteins and was reversibly inhibited. Ceruloplasmin was the predominant protein associated with myeloperoxidase. When the purified proteins were mixed, they became strongly but reversibly associated. Ceruloplasmin was a potent inhibitor of purified myeloperoxidase, inhibiting production of hypochlorous acid by 50% at 25 nm. Ceruloplasmin rapidly reduced Compound I, the FeV redox intermediate of myeloperoxidase, to Compound II, which has FeIV in its heme prosthetic groups. It also prevented the fast reduction of Compound II by tyrosine. In the presence of chloride and hydrogen peroxide, ceruloplasmin converted myeloperoxidase to Compound II and slowed its conversion back to the ferric enzyme. Collectively, our results indicate that ceruloplasmin inhibits myeloperoxidase by reducing Compound I and then trapping the enzyme as inactive Compound II. We propose that ceruloplasmin should provide a protective shield against inadvertent oxidant production by myeloperoxidase during inflammation. PMID:23306200
Aluminum ammonium sulfate dodecahydrate purified from traditional Chinese medicinal herb Korean monkshood root is a potent matrix metalloproteinase inhibitor.

PubMed

Shen, Yehua; Liu, Sen; Jin, Fenghai; Mu, Tianyang; Li, Cong; Jiang, Kun; Tian, Weihua; Yu, Dahai; Zhang, Yingqi; Fang, Xuexun

2012-06-01

Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases and key regulators for many physiological and pathological functions. The MMP inhibitors have been shown to modulate diseases such as cancer, inflammation, and cardiovascular diseases. In this paper we tracked the MMP inhibitory activities of the traditional Chinese medicinal herb Korean Monkshood Root. The purified active ingredient was identified by the elemental analysis, infrared spectrum (IR) and X-ray diffraction as aluminum ammonium sulfate dodecahydrate. This inorganic compound showed inhibitory activities toward a number of MMP family members. In particular, it has a strong inhibitory effect toward MMP-2 and MMP-9, with IC50 values of 0.54 and 0.50 μM, respectively. Further analysis suggested that the MMP inhibitory activity is mainly due to Al(3+). Cell viability assays using human fibrosarcoma HT1080 cells showed aluminum ammonium sulfate had minimal cyto-toxicity with a concentration up to 500 μM. However, within 50 μM, it exhibited significant inhibition of cell invasion. To our knowledge, there has been no previous report of inorganic form of the MMP inhibitor with strong inhibitory activity. Our results for the first time showed that aluminum ammonium sulfate is an inorganic form of MMP inhibitor with high potency, and can be used to interfere with MMP related cellular processes.
In vitro formation of recycling vesicles from endosomes requires adaptor protein-1/clathrin and is regulated by rab4 and the connector rabaptin-5.

PubMed

Pagano, Adriana; Crottet, Pascal; Prescianotto-Baschong, Cristina; Spiess, Martin

2004-11-01

The involvement of clathrin and associated adaptor proteins in receptor recycling from endosomes back to the plasma membrane is controversial. We have used an in vitro assay to identify the molecular requirements for the formation of recycling vesicles. Cells expressing the asialoglycoprotein receptor H1, a typical recycling receptor, were surface biotinylated and then allowed to endocytose for 10 min. After stripping away surface-biotin, the cells were permeabilized and the cytosol washed away. In a temperature-, cytosol-, and nucleotide-dependent manner, the formation of sealed vesicles containing biotinylated H1 could be reconstituted. Vesicle formation was strongly inhibited upon immunodepletion of adaptor protein (AP)-1, but not of AP-2 or AP-3, from the cytosol, and was restored by readdition of purified AP-1. Vesicle formation was stimulated by supplemented clathrin, but inhibited by brefeldin A, consistent with the involvement of ARF1 and a brefeldin-sensitive guanine nucleotide exchange factor. The GTPase rab4, but not rab5, was required to generate endosome-derived vesicles. Depletion of rabaptin-5/rabex-5, a known interactor of both rab4 and gamma-adaptin, stimulated and addition of the purified protein strongly inhibited vesicle production. The results indicate that recycling is mediated by AP-1/clathrin-coated vesicles and regulated by rab4 and rabaptin-5/rabex-5.
Foundational errors in the Neutral and Nearly-Neutral theories of evolution in relation to the Synthetic Theory: is a new evolutionary paradigm necessary?

PubMed

Valenzuela, Carlos Y

2013-01-01

The Neutral Theory of Evolution (NTE) proposes mutation and random genetic drift as the most important evolutionary factors. The most conspicuous feature of evolution is the genomic stability during paleontological eras and lack of variation among taxa; 98% or more of nucleotide sites are monomorphic within a species. NTE explains this homology by random fixation of neutral bases and negative selection (purifying selection) that does not contribute either to evolution or polymorphisms. Purifying selection is insufficient to account for this evolutionary feature and the Nearly-Neutral Theory of Evolution (N-NTE) included negative selection with coefficients as low as mutation rate. These NTE and N-NTE propositions are thermodynamically (tendency to random distributions, second law), biotically (recurrent mutation), logically and mathematically (resilient equilibria instead of fixation by drift) untenable. Recurrent forward and backward mutation and random fluctuations of base frequencies alone in a site make life organization and fixations impossible. Drift is not a directional evolutionary factor, but a directional tendency of matter-energy processes (second law) which threatens the biotic organization. Drift cannot drive evolution. In a site, the mutation rates among bases and selection coefficients determine the resilient equilibrium frequency of bases that genetic drift cannot change. The expected neutral random interaction among nucleotides is zero; however, huge interactions and periodicities were found between bases of dinucleotides separated by 1, 2... and more than 1,000 sites. Every base is co-adapted with the whole genome. Neutralists found that neutral evolution is independent of population size (N); thus neutral evolution should be independent of drift, because drift effect is dependent upon N. Also, chromosome size and shape as well as protein size are far from random.
Nucleotide selectivity defect and mutator phenotype conferred by a colon cancer-associated DNA polymerase δ mutation in human cells

PubMed Central

Mertz, Tony M.; Baranovskiy, Andrey G.; Wang, Jing; Tahirov, Tahir H.; Shcherbakova, Polina V.

2017-01-01

Mutations in the POLD1 and POLE genes encoding DNA polymerases δ (Polδ) and ε (Polε) cause hereditary colorectal cancer (CRC) and have been found in many sporadic colorectal and endometrial tumors. Much attention has been focused on POLE exonuclease domain mutations, which occur frequently in hypermutated DNA mismatch repair (MMR)-proficient tumors and appear to be responsible for the bulk of genomic instability in these tumors. In contrast, somatic POLD1 mutations are seen less frequently and typically occur in MMR-deficient tumors. Their functional significance is often unclear. Here we demonstrate that expression of the cancer-associated POLD1-R689W allele is strongly mutagenic in human cells. The mutation rate increased synergistically when the POLD1-R689W expression was combined with a MMR defect, indicating that the mutator effect of POLD1-R689W results from a high rate of replication errors. Purified human Polδ-R689W has normal exonuclease activity, but the nucleotide selectivity of the enzyme is severely impaired, providing a mechanistic explanation for the increased mutation rate in the POLD1-R689W-expressing cells. The vast majority of mutations induced by the POLD1-R689W are GC→TA transversions and GC→AT transitions, with transversions showing a strong strand bias and a remarkable preference for polypurine/polypyrimidine sequences. The mutational specificity of the Polδ variant matches that of the hypermutated CRC cell line, HCT15, in which this variant was first identified. The results provide compelling evidence for the pathogenic role of the POLD1-R689W mutation in the development of the human tumor and emphasize the need to experimentally determine the significance of Polδ variants present in sporadic tumors. PMID:28368425
Human Variation in Short Regions Predisposed to Deep Evolutionary Conservation

PubMed Central

Loots, Gabriela G.; Ovcharenko, Ivan

2010-01-01

The landscape of the human genome consists of millions of short islands of conservation that are 100% conserved across multiple vertebrate genomes (termed “bricks”), the majority of which are located in noncoding regions. Several hundred thousand bricks are deeply conserved reaching the genomes of amphibians and fish. Deep phylogenetic conservation of noncoding DNA has been reported to be strongly associated with the presence of gene regulatory elements, introducing bricks as a proxy to the functional noncoding landscape of the human genome. Here, we report a significant overrepresentation of bricks in the promoters of transcription factors and developmental genes, where the high level of phylogenetic conservation correlates with an increase in brick overrepresentation. We also found that the presence of a brick dictates a predisposition to evolutionary constraint, with only 0.7% of the amniota brick central nucleotides being diverged within the primate lineage—an 11-fold reduction in the divergence rate compared with random expectation. Human single-nucleotide polymorphism (SNP) data explains only 3% of primate-specific variation in amniota bricks, thus arguing for a widespread fixation of brick mutations within the primate lineage and prior to human radiation. This variation, in turn, might have been utilized as a driving force for primate- and hominoid-specific adaptation. We also discovered a pronounced deviation from the evolutionary predisposition in the human lineage, with over 20-fold increase in the substitution rate at brick SNP sites over expected values. In addition, contrary to typical brick mutations, brick variation commonly encountered in the human population displays limited, if any, signatures of negative selection as measured by the minor allele frequency and population differentiation (F-statistical measure) measures. These observations argue for the plasticity of gene regulatory mechanisms in vertebrates—with evidence of strong purifying selection acting on the gene regulatory landscape of the human genome, where widespread advantageous mutations in putative regulatory elements are likely utilized in functional diversification and adaptation of species. PMID:20093432
Hydrolysis Rates of Different Small Interfering RNAs (siRNAs) by the RNA Silencing Promoter Complex, C3PO, Determines Their Regulation by Phospholipase Cβ*

PubMed Central

Sahu, Shriya; Philip, Finly; Scarlata, Suzanne

2014-01-01

C3PO plays a key role in promoting RNA-induced gene silencing. C3PO consists of two subunits of the endonuclease translin-associated factor X (TRAX) and six subunits of the nucleotide-binding protein translin. We have found that TRAX binds strongly to phospholipase Cβ (PLCβ), which transmits G protein signals from many hormones and sensory inputs. The association between PLCβ and TRAX is thought to underlie the ability of PLCβ to reverse gene silencing by small interfering RNAs. However, this reversal only occurs for some genes (e.g. GAPDH and LDH) but not others (e.g. Hsp90 and cyclophilin A). To understand this specificity, we carried out studies using fluorescence-based methods. In cells, we find that PLCβ, TRAX, and their complexes are identically distributed through the cytosol suggesting that selectivity is not due to large scale sequestration of either the free or complexed proteins. Using purified proteins, we find that PLCβ binds ∼5-fold more weakly to translin than to TRAX but ∼2-fold more strongly to C3PO. PLCβ does not alter TRAX-translin assembly to C3PO, and brightness studies suggest one PLCβ binds to one C3PO octamer without a change in the number of TRAX/translin molecules suggesting that PLCβ binds to an external site. Functionally, we find that C3PO hydrolyzes siRNA(GAPDH) at a faster rate than siRNA(Hsp90). However, when PLCβ is bound to C3PO, the hydrolysis rate of siRNA(GAPDH) becomes comparable with siRNA(Hsp90). Our results show that the selectivity of PLCβ toward certain genes lies in the rate at which the RNA is hydrolyzed by C3PO. PMID:24338081
Preparation and testing of a Haemophilus influenzae Type b/Hepatitis B surface antigen conjugate vaccine.

PubMed

An, So Jung; Woo, Joo Sung; Chae, Myung Hwa; Kothari, Sudeep; Carbis, Rodney

2015-03-24

The majority of conjugate vaccines focus on inducing an antibody response to the polysaccharide antigen and the carrier protein is present primarily to induce a T-cell dependent response. In this study conjugates consisting of poly(ribosylribitolphosphate) (PRP) purified from Haemophilus influenzae Type b bound to Hepatitis B virus surface antigen (HBsAg) virus like particles were prepared with the aim of inducing an antibody response to not only the PRP but also the HBsAg. A conjugate consisting of PRP bound to HBsAg via an adipic acid dihydrazide (ADH) spacer induced strong IgG antibodies to both the PRP and HBsAg. When conjugation was performed without the ADH spacer the induction of an anti-PRP response was equivalent to that seen by conjugate with the ADH spacer, however, a negligible anti-HBsAg response was induced. For comparison, PRP was conjugated to diphtheria toxoid (DT) and Vi polysaccharide purified from Salmonella Typhi conjugated to HBsAg both using an ADH spacer. The PRPAH-DT conjugate induced strong anti-PRP and anti-DT responses, the Vi-AHHBsAg conjugate induced a good anti-HBsAg response but not as strong as that induced by the PRPAH-HBsAg conjugate. This study demonstrated that in mice it was possible to induce robust antibody responses to both polysaccharide and carrier protein provided the conjugate has certain physico-chemical properties. A PRPAH-HBsAg conjugate with the capacity to induce anti-PRP and anti-HBsAg responses could be incorporated into a multivalent pediatric vaccine and simplify formulation of such a vaccine. Copyright © 2015 Elsevier Ltd. All rights reserved.
A nanobody directed to a functional epitope on VEGF, as a novel strategy for cancer treatment

DOE Office of Scientific and Technical Information (OSTI.GOV)

Farajpour, Zahra; Rahbarizadeh, Fatemeh, E-mail: rahbarif@modares.ac.ir; Kazemi, Bahram

Highlights: • A novel nanobody directed to antigenic regions on VEGF was identified. • Our nanobody was successfully purified. • Our nanobody significantly inhibited VEGF-induced proliferation of HUVECs in a dose dependent manner. - Abstract: Compelling evidence suggests that vascular endothelial growth factor (VEGF), due to its essential role in angiogenesis, is a critical target for cancer treatment. Neutralizing monoclonal antibodies against VEGF are important class of drugs used in cancer therapy. However, the cost of production, large size, and immunogenicity are main drawbacks of conventional monoclonal therapy. Nanobodies are the smallest antigen-binding antibody fragments, which occur naturally in camelidae.more » Because of their remarkable features, we decided to use an immune library of nanobody to direct phage display to recognition of novel functional epitopes on VEGF. Four rounds of selection were performed and six phage-displayed nanobodies were obtained from an immune phage library. The most reactive clone in whole-cell ELISA experiments, was purified and assessed in proliferation inhibition assay. Purified ZFR-5 not only blocked interaction of VEGF with its receptor in cell ELISA experiments, but also was able to significantly inhibit proliferation response of human umbilical vein endothelial cells to VEGF in a dose-dependent manner. Taken together, our study demonstrates that by using whole-cell ELISA experiments, nanobodies against antigenic regions included in interaction of VEGF with its receptors can be directed. Because of unique and intrinsic properties of a nanobody and the ability of selected nanobody for blocking the epitope that is important for biological function of VEGF, it represents novel potential drug candidate.« less
A robust robotic high-throughput antibody purification platform.

PubMed

Schmidt, Peter M; Abdo, Michael; Butcher, Rebecca E; Yap, Min-Yin; Scotney, Pierre D; Ramunno, Melanie L; Martin-Roussety, Genevieve; Owczarek, Catherine; Hardy, Matthew P; Chen, Chao-Guang; Fabri, Louis J

2016-07-15

Monoclonal antibodies (mAbs) have become the fastest growing segment in the drug market with annual sales of more than 40 billion US$ in 2013. The selection of lead candidate molecules involves the generation of large repertoires of antibodies from which to choose a final therapeutic candidate. Improvements in the ability to rapidly produce and purify many antibodies in sufficient quantities reduces the lead time for selection which ultimately impacts on the speed with which an antibody may transition through the research stage and into product development. Miniaturization and automation of chromatography using micro columns (RoboColumns(®) from Atoll GmbH) coupled to an automated liquid handling instrument (ALH; Freedom EVO(®) from Tecan) has been a successful approach to establish high throughput process development platforms. Recent advances in transient gene expression (TGE) using the high-titre Expi293F™ system have enabled recombinant mAb titres of greater than 500mg/L. These relatively high protein titres reduce the volume required to generate several milligrams of individual antibodies for initial biochemical and biological downstream assays, making TGE in the Expi293F™ system ideally suited to high throughput chromatography on an ALH. The present publication describes a novel platform for purifying Expi293F™-expressed recombinant mAbs directly from cell-free culture supernatant on a Perkin Elmer JANUS-VariSpan ALH equipped with a plate shuttle device. The purification platform allows automated 2-step purification (Protein A-desalting/size exclusion chromatography) of several hundred mAbs per week. The new robotic method can purify mAbs with high recovery (>90%) at sub-milligram level with yields of up to 2mg from 4mL of cell-free culture supernatant. Copyright © 2016 Elsevier B.V. All rights reserved.

Purification and biochemical heterogeneity of the mammalian SWI-SNF complex.

PubMed Central

Wang, W; Côté, J; Xue, Y; Zhou, S; Khavari, P A; Biggar, S R; Muchardt, C; Kalpana, G V; Goff, S P; Yaniv, M; Workman, J L; Crabtree, G R

1996-01-01

We have purified distinct complexes of nine to 12 proteins [referred to as BRG1-associated factors (BAFs)] from several mammalian cell lines using an antibody to the SWI2-SNF2 homolog BRG1. Microsequencing revealed that the 47 kDa BAF is identical to INI1. Previously INI1 has been shown to interact with and activate human immunodeficiency virus integrase and to be homologous to the yeast SNF5 gene. A group of BAF47-associated proteins were affinity purified with antibodies against INI1/BAF47 and were found to be identical to those co-purified with BRG1, strongly indicating that this group of proteins associates tightly and is likely to be the mammalian equivalent of the yeast SWI-SNF complex. Complexes containing BRG1 can disrupt nucleosomes and facilitate the binding of GAL4-VP16 to a nucleosomal template similar to the yeast SWI-SNF complex. Purification of the complex from several cell lines demonstrates that it is heterogeneous with respect to subunit composition. The two SWI-SNF2 homologs, BRG1 and hbrm, were found in separate complexes. Certain cell lines completely lack BRG1 and hbrm, indicating that they are not essential for cell viability and that the mammalian SWI-SNF complex may be tailored to the needs of a differentiated cell type. Images PMID:8895581
Collagenase produced from Aspergillus sp. (UCP 1276) using chicken feather industrial residue.

PubMed

Ferreira, Catarina Michelle Oliveira; Correia, Patyanne Carvalho; Brandão-Costa, Romero Marcos Pedrosa; Albuquerque, Wendell Wagner Campos; Lin Liu, Tatiana Pereira Shin; Campos-Takaki, Galba Maria; Porto, Ana Lúcia Figueiredo

2017-05-01

An extracellular collagenolytic serine protease was purified from Aspergillus sp., isolated from the Caatinga biome in northeast Brazil by a two-step chromatographic procedure, using an anion-exchanger and gel filtration. The enzyme was produced by submerged fermentation of feather residue as a substrate. The purified collagenase showed a 2.09-fold increase in specific activity and 22.85% yield. The enzyme was a monomeric protein with a molecular mass of 28.7 kDa, estimated by an SDS-PAGE and AKTA system. The optimum temperature and pH for enzyme activity were around 40°C and pH 8.0, respectively. The enzyme was strongly inhibited by phenyl-methylsulfonyl fluoride, a serine protease inhibitor, and was thermostable until 65°C for 1 h. We then evaluated the enzyme's potential for degradation of Type I and Type V collagens for producing peptides with antifungal activity. Our results revealed that the cleavage of Type V collagen yielded more effective peptides than Type I, inhibiting growth of Aspergillus terreus, Aspergillus japonicus and Aspergillus parasiticus. Both groups of peptides (Type I and Type V) were identified by SDS-PAGE. To conclude, the thermostable collagenase we purified in this study has various potentially useful applications in the fields of biochemistry, biotechnology and biomedical sciences. Copyright © 2016 John Wiley & Sons, Ltd.
Effects of the lipid environment, cholesterol and bile acids on the function of the purified and reconstituted human ABCG2 protein.

PubMed

Telbisz, Ágnes; Özvegy-Laczka, Csilla; Hegedűs, Tamás; Váradi, András; Sarkadi, Balázs

2013-03-01

The human ABCG2 multidrug transporter actively extrudes a wide range of hydrophobic drugs and xenobiotics recognized by the transporter in the membrane phase. In order to examine the molecular nature of the transporter and its effects on the lipid environment, we have established an efficient protocol for the purification and reconstitution of the functional protein. We found that the drug-stimulated ATPase and the transport activity of ABCG2 are fully preserved by applying excess lipids and mild detergents during solubilization, whereas a detergent-induced dissociation of the ABCG2 dimer causes an irreversible inactivation. By using the purified and reconstituted protein we demonstrate that cholesterol is an essential activator, whereas bile acids are important modulators of ABCG2 activity. Both wild-type ABCG2 and its R482G mutant variant require cholesterol for full activity, although they exhibit different cholesterol sensitivities. Bile acids strongly decrease the basal ABCG2-ATPase activity both in the wild-type ABCG2 and in the mutant variant. These data reinforce the results for the modulatory effects of cholesterol and bile acids of ABCG2 investigated in a complex cell membrane environment. Moreover, these experiments open the possibility to perform functional and structural studies with a purified, reconstituted and highly active ABCG2 multidrug transporter.
Purification and characterization of extracellular lipase from a new strain: Pseudomonas aeruginosa SRT 9

PubMed Central

Borkar, Prita S.; Bodade, Ragini G.; Rao, Srinivasa R.; Khobragade, C.N.

2009-01-01

An extra cellular lipase was isolated and purified from the culture broth of Pseudomonas aeruginosa SRT 9 to apparent homogeneity using ammonium sulfate precipitation followed by chromatographic techniques on phenyl Sepharose CL- 4B and Mono Q HR 5/5 column, resulting in a purification factor of 98 fold with specific activity of 12307.8 U/mg. The molecular weight of the purified lipase was estimated by SDS-PAGE to be 29 kDa with isoelectric point of 4.5. Maximum lipase activity was observed in a wide range of temperature and pH values with optimum temperature of 55ºC and pH 6.9. The lipase preferably acted on triacylglycerols of long chain (C14-C16) fatty acids. The lipase was inhibited strongly by EDTA suggesting the enzyme might be metalloprotein. SDS and metal ions such as Hg2+, Zn2+, Cu2+, Ag2+ and Fe2+ decreased the lipase activity remarkedly. Its marked stability and activity in organic solvents suggest that this lipase is highly suitable as a biotechnological tool with a variety of applications including organo synthetic reactions and preparation of enantiomerically pure pharmaceuticals. The Km and Vmax value of the purified enzyme for triolein hydrolysis were calculated to be 1.11 mmol/L and 0.05 mmol/L/min respectively. PMID:24031373
Cowpea ribonuclease: properties and effect of NaCl-salinity on its activation during seed germination and seedling establishment.

PubMed

Gomes-Filho, Enéas; Lima, Carmen Rogélia Farias Machado; Costa, José Hélio; da Silva, Ana Cláudia Marinho; da Guia Silva Lima, Maria; de Lacerda, Claudivan Feitosa; Prisco, José Tarquinio

2008-01-01

Pitiúba cowpea [Vigna unguiculata (L.) Walp] seeds were germinated in distilled water (control treatment) or in 100 mM NaCl solution (salt treatment), and RNase was purified from different parts of the seedlings. Seedling growth was reduced by the NaCl treatment. RNase activity was low in cotyledons of quiescent seeds, but the enzyme was activated during germination and seedling establishment. Salinity reduced cotyledon RNase activity, and this effect appeared to be due to a delay in its activation. The RNases from roots, stems, and leaves were immunologically identical to that found in cotyledons. Partially purified RNase fractions from the different parts of the seedling showed some activity with DNA as substrate. However, this DNA hydrolyzing activity was much lower than that of RNA hydrolyzing activity. The DNA hydrolyzing activity was strongly inhibited by Cu(2+), Hg(2+), and Zn(2+) ions, stimulated by MgCl(2), and slowly inhibited by EDTA. This activity from the most purified fraction was inhibited by increasing concentrations of RNA in the reaction medium. It is suggested that the major biological role of this cotyledon RNase would be to hydrolyze seed storage RNA during germination and seedling establishment, and it was discussed that it might have a protective role against abiotic stress during later part of seedling establishment.
Molecular homogeneity of heat-stable enterotoxins produced by bovine enterotoxigenic Escherichia coli.

PubMed Central

Saeed, A M; Magnuson, N S; Sriranganathan, N; Burger, D; Cosand, W

1984-01-01

Heat-stable enterotoxins (STs) from four strains of bovine enterotoxigenic Escherichia coli representing four serogroups were purified to homogeneity by utilizing previously published purification schemata. Biochemical characterization of the purified STs showed that they met the basic criteria for the heat-stable enterotoxins of E. coli. Amino acid analysis of the purified STs revealed that they were peptides of identical amino acid composition. This composition consisted of 18 residues of 10 different amino acids, 6 of which were cysteine. The amino acid composition of the four ST peptides was identical to that reported for the STs of human and porcine E. coli. In addition, complete sequence analysis of two of the ST peptides and partial sequencing of several others revealed strong homology to the sequences of STs from human and porcine E. coli and to the sequence predicted from the last 18 codons of the transposon Tn1681. There was also substantial homology to the sequence predicted from the ST-coding genetic element of human E. coli, which may indicate the existence of identical bioactive configuration among ST peptides of E. coli strains of various host origins. These data support the hypothesis that STs produced by human, bovine, and porcine E. coli are coded by a closely related genetic element which may have originated from a single, widely disseminated transposon. Images PMID:6376355
[Antimicrobial effect on some zoonotic bacteria, of the cell-free fermentation fluid and purified peptide fraction of the entomopathogenic bacterium, Xenorhabdus budapestensis].

PubMed

Burgettiné Böszörményi, Erzsébet; Barcs, István; Domján, Gyula; Bélafiné Bakó, Katalin; Fodor, András; Makrai, László; Vozik, Dávid

2015-11-01

Many multi-resistant patogens appear continuously resulting in a permanent need for the development of novel antibiotics. A large number of antibiotics introduced in clinical and veterinary practices are not effective. Antibacterial peptides with unusual mode of action may represent a promising option against multi-resistant pathogens. The entomopathogenic Xenorhabdus budapestensis bacteria produce several different antimicrobial peptides compounds such as bicornutin-A and fabclavin. The aim of the authors was to evaluate the in vitro antibacterial effect of Xenorhabdus budapestensis using zoonotic patogen bacteria. Cell-free conditioned media and purified peptide fractions of Xenorhabdus budapestensis were tested on Gram-positive (Rhodococcus equi, Erysipelothrix rhusiopathia, Staphylococcus aureus, Streptococcus equi, Corynebacterium pseudotuberculosis, Listeria monocytagenes) and Gram-negative bacteria (Salmonella gallinarum, Salmonella derbi, Bordatella bronchoseptica, Escherichia coli, Pasteurella multocida, Aeromonas hydrophila) using agar diffusion test on blood agar plates. It was found that Xenorhabdus budapestensis bacteria produced compounds with strong and dose-dependent effects on the tested organisms. Purified peptid fraction exerted a more marked effect than cell free conditioned media. Gram-positive bacteria were more sensitive to this antibacterial effect than Gram-negative bacteria. Antibacterial peptide compound from Xenorhabdus budapestensis exert marked antibacterial effect on zoonotic patogen bacteria and they should be further evaluated in future for their potential use in the control or prevention of zoonoses.
Antibody Characterization Process | Office of Cancer Clinical Proteomics Research

Cancer.gov

The goal of the NCI's Antibody Characterization Program (ACP) is to have three monoclonal antibodies produced for each successfully expressed/purified recombinant antigen and one antibody per peptide (1 to 3 peptides per protein). To date, over 4000 clones have been screened before selecting the current 393 antibodies. They are winnowed down based on the projected end use of the antibody.
Antimalarial drug discovery: screening of Brazilian medicinal plants and purified compounds.

PubMed

Krettli, Antoniana Ursine

2009-02-01

Malaria is the most important parasitic disease and its control depends on specific chemotherapy, now complicated by Plasmodium falciparum that has become resistant to most commonly available antimalarials. Treatment of the disease requires quinine or drug combinations of artemisinin derivatives and other antimalarials. Further drug resistance is expected. New active compounds need to be discovered. To find new antimalarials from medicinal and randomly collected plants, crude extracts are screened against P. falciparum in cultures and in malaria animal models, following bioassays of purified fractions, and cytotoxicity tests. For antimalarial research, screening medicinal plants is more efficient than screening randomly chosen plants. Biomonitored fractionation allows selection of new active molecules identified as potential antimalarials in multidisciplinary projects in Brazil; no new molecule is available for human testing. The advantages of projects based on ethnopharmacology are discussed.
Anti-tumour potential of a gallic acid-containing phenolic fraction from Oenothera biennis.

PubMed

Pellegrina, Chiara Dalla; Padovani, Giorgia; Mainente, Federica; Zoccatelli, Gianni; Bissoli, Gaetano; Mosconi, Silvia; Veneri, Gianluca; Peruffo, Angelo; Andrighetto, Giancarlo; Rizzi, Corrado; Chignola, Roberto

2005-08-08

A phenolic fraction purified form defatted seeds of Oenothera biennis promoted selective apoptosis of human and mouse bone marrow-derived cell lines following first-order kinetics through a caspase-dependent pathway. In non-leukemia tumour cell lines, such as human colon carcinoma CaCo(2) cells and mouse fibrosarcoma WEHI164 cells, this fraction inhibited (3)H-thymidine incorporation but not cell death or cell cycle arrest. Human peripheral blood mononuclear cells showed low sensitivity to treatment. Single bolus injection of the phenolic fraction could delay the growth of established myeloma tumours in syngeneic animals. HPLC and mass spectrometry analysis revealed that the fraction contains gallic acid. However, the biological activity of the fraction differs from the activity of this phenol and hence it should be attributed to other co-purified molecules which remain still unidentified.
Process for forming a nickel foil with controlled and predetermined permeability to hydrogen

DOEpatents

Engelhaupt, Darell E.

1981-09-22

The present invention provides a novel process for forming a nickel foil having a controlled and predetermined hydrogen permeability. This process includes the steps of passing a nickel plating bath through a suitable cation exchange resin to provide a purified nickel plating bath free of copper and gold cations, immersing a nickel anode and a suitable cathode in the purified nickel plating bath containing a selected concentration of an organic sulfonic acid such as a napthalene-trisulfonic acid, electrodepositing a nickel layer having the thickness of a foil onto the cathode, and separating the nickel layer from the cathode to provide a nickel foil. The anode is a readily-corrodible nickel anode. The present invention also provides a novel nickel foil having a greater hydrogen permeability than palladium at room temperature.
Anti-Legionella activity of staphylococcal hemolytic peptides.

PubMed

Marchand, A; Verdon, J; Lacombe, C; Crapart, S; Héchard, Y; Berjeaud, J M

2011-05-01

A collection of various Staphylococci was screened for their anti-Legionella activity. Nine of the tested strains were found to secrete anti-Legionella compounds. The culture supernatants of the strains, described in the literature to produce hemolytic peptides, were successfully submitted to a two step purification process. All the purified compounds, except one, corresponded to previously described hemolytic peptides and were not known for their anti-Legionella activity. By comparison of the minimal inhibitory concentrations, minimal permeabilization concentrations, decrease in the number of cultivable bacteria, hemolytic activity and selectivity, the purified peptides could be separated in two groups. First group, with warnericin RK as a leader, corresponds to the more hemolytic and bactericidal peptides. The peptides of the second group, represented by the PSMα from Staphylococcus epidermidis, appeared bacteriostatic and poorly hemolytic. Copyright © 2011 Elsevier Inc. All rights reserved.
Induction of specific immune unresponsiveness with purified mixed leukocyte culture-activated T lymphoblasts as autoimmunogen. II. An analysis of the effects measured at the cellular and serological levels

PubMed Central

1978-01-01

T lymphoblasts specific for foreign histocompatibility antigens and purified via mixed leukocyte culture (MLC) and 1 g velocity sedimentation procedures can be used as autoimmunogen to produce specific immunological unresponsiveness in adult animals. This unresponsiveness is positively correlated to the production of autoanti- idiotypic antibodies in the blast immunized animals and no evidence of coexisting alloimmunity was found. We consider this autoanti-idiotypic immunity to be the specific inducing agent of the immune tolerance. The blast immunization procedure will lead to selective reduction in T-cell reactivity against the relevant alloantigens as measured by MLC, cell- mediated lympholysis, or graft-versus-host assays. However, in individual animals, dichtomy in suppression between two T-cell assays could sometimes be observed indicating elimination of only a select group of idiotypic functionally distinct population of T cells in these blast-immunized animals. Attempts to abrogate already immune animals by the autoblast procedure were successful, in part suggesting the use of the present procedure when trying to induce in accelerated reversion of such immunity. PMID:75235
Accumulation of slightly deleterious mutations in the mitochondrial genome: a hallmark of animal domestication.

PubMed

Hughes, Austin L

2013-02-15

The hypothesis that domestication leads to a relaxation of purifying selection on mitochondrial (mt) genomes was tested by comparative analysis of mt genes from dog, pig, chicken, and silkworm. The three vertebrate species showed mt genome phylogenies in which domestic and wild isolates were intermingled, whereas the domestic silkworm (Bombyx mori) formed a distinct cluster nested within its closest wild relative (Bombyx mandarina). In spite of these differences in phylogenetic pattern, significantly greater proportions of nonsynonymous SNPs than of synonymous SNPs were unique to the domestic populations of all four species. Likewise, in all four species, significantly greater proportions of RNA-encoding SNPs than of synonymous SNPs were unique to the domestic populations. Thus, domestic populations were characterized by an excess of unique polymorphisms in two categories generally subject to purifying selection: nonsynonymous sites and RNA-encoding sites. Many of these unique polymorphisms thus seem likely to be slightly deleterious; the latter hypothesis was supported by the generally lower gene diversities of polymorphisms unique to domestic populations in comparison to those of polymorphisms shared by domestic and wild populations. Copyright © 2012 Elsevier B.V. All rights reserved.
An integrated map of genetic variation from 1,092 human genomes

PubMed Central

2012-01-01

Summary Through characterising the geographic and functional spectrum of human genetic variation, the 1000 Genomes Project aims to build a resource to help understand the genetic contribution to disease. We describe the genomes of 1,092 individuals from 14 populations, constructed using a combination of low-coverage whole-genome and exome sequencing. By developing methodologies to integrate information across multiple algorithms and diverse data sources we provide a validated haplotype map of 38 million SNPs, 1.4 million indels and over 14 thousand larger deletions. We show that individuals from different populations carry different profiles of rare and common variants and that low-frequency variants show substantial geographic differentiation, which is further increased by the action of purifying selection. We show that evolutionary conservation and coding consequence are key determinants of the strength of purifying selection, that rare-variant load varies substantially across biological pathways and that each individual harbours hundreds of rare non-coding variants at conserved sites, such as transcription-factor-motif disrupting changes. This resource, which captures up to 98% of accessible SNPs at a frequency of 1% in populations of medical genetics focus, enables analysis of common and low-frequency variants in individuals from diverse, including admixed, populations. PMID:23128226
Production and Characterization of a New α-Glucosidase Inhibitory Peptide from Aspergillus oryzae N159-1

PubMed Central

Kang, Min-Gu; Yi, Sung-Hun

2013-01-01

An α-glucosidase inhibitor was developed from Aspergillus oryzae N159-1, which was screened from traditional fermented Korean foods. The intracellular concentration of the inhibitor reached its highest level when the fungus was cultured in tryptic soy broth medium at 27℃ for five days. The inhibitor was purified using a series of purification steps involving ultrafiltration, Sephadex G-25 gel permeation chromatography, strong cation exchange solid phase extraction, reverse-phase high performance liquid chromatography, and size exclusion chromatography. The final yield of the purification was 1.9%. Results of the liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis indicated that the purified α-glucosidase inhibitor was a tri-peptide, Pro-Phe-Pro, with the molecular weight of 360.1 Da. The IC50 value of the peptide against α-glucosidase activity was 3.1 mg/mL. Using Lineweaver-Burk plot analysis, the inhibition pattern indicated that the inhibitor acts as a mixed type inhibitor. PMID:24198670
Analysis of the axial filaments of Treponema hyodysenteriae by SDS-PAGE and immunoblotting.

PubMed

Kent, K A; Sellwood, R; Lemcke, R M; Burrows, M R; Lysons, R J

1989-06-01

Purified axial filaments from eight serotypes of Treponema hyodysenteriae and two non-pathogenic intestinal spirochaetes were characterized by SDS-PAGE and Western blotting. Axial filaments of all ten strains had similar SDS-PAGE profiles; five major axial filament polypeptides were identified, with molecular masses of 43.8, 38, 34.8, 32.8 and 29.4 kDa. Hyperimmune gnotobiotic pig serum raised against purified axial filaments of strain P18A (serotype 4) cross-reacted with all other serotypes and with the non-pathogens, and convalescent serum taken from a pig with persistent swine dysentery also showed a strong response to the axial filament polypeptides. Hyperimmune gnotobiotic pig serum raised against axial filaments failed to agglutinate viable organisms and did not inhibit growth in vitro. Hence, the axial filaments of T. hyodysenteriae have been identified as major immunodominant antigens, although the role that antibodies to these antigens play in protection has yet to be established.
LARGE SCALE METHOD FOR THE PRODUCTION AND PURIFICATION OF CURIUM

DOEpatents

Higgins, G.H.; Crane, W.W.T.

1959-05-19

A large-scale process for production and purification of Cm/sup 242/ is described. Aluminum slugs containing Am are irradiated and declad in a NaOH-- NaHO/sub 3/ solution at 85 to 100 deg C. The resulting slurry filtered and washed with NaOH, NH/sub 4/OH, and H/sub 2/O. Recovery of Cm from filtrate and washings is effected by an Fe(OH)/sub 3/ precipitation. The precipitates are then combined and dissolved ln HCl and refractory oxides centrifuged out. These oxides are then fused with Na/sub 2/CO/sub 3/ and dissolved in HCl. The solution is evaporated and LiCl solution added. The Cm, rare earths, and anionic impurities are adsorbed on a strong-base anfon exchange resin. Impurities are eluted with LiCl--HCl solution, rare earths and Cm are eluted by HCl. Other ion exchange steps further purify the Cm. The Cm is then precipitated as fluoride and used in this form or further purified and processed. (T.R.H.)
Generation of 2-Furfurylthiol by Carbon-Sulfur Lyase from the Baijiu Yeast Saccharomyces cerevisiae G20.

PubMed

Zha, Musu; Sun, Baoguo; Yin, Sheng; Mehmood, Arshad; Cheng, Lei; Wang, Chengtao

2018-03-07

2-Furfurylthiol is the representative aroma compound of Chinese sesame-flavored baijiu. Previous studies demonstrated that baijiu yeasts could generate 2-furfurylthiol using furfural and l-cysteine as precursors and that the Saccharomyces cerevisiae genes STR3 and CYS3 are closely related to 2-furfurylthiol biosynthesis. To confirm the mechanism of the STR3- and CYS3-gene products on 2-furfurylthiol biosynthesis, their encoded proteins were purified, and we confirmed their activities as carbon-sulfur lyases. Str3p and Cys3p were able to cleave the cysteine-furfural conjugate to release 2-furfurylthiol. Moreover, the characterization of the enzymatic properties of the purified proteins shows good thermal stabilities and wide pH tolerances, which enable their strong potential for various applications. These data provide direct evidence that yeast Str3p and Cys3p release 2-furfurylthiol in vitro, which can be applied to improve baijiu flavor.
The Adsorption Capacity of GONs/CMC/Fe3O4 Magnetic Composite Microspheres and Applications for Purifying Dye Wastewater

PubMed Central

Lv, Shenghua; Zhu, Linlin; Li, Ying; Jia, Chunmao; Sun, Shiyu

2017-01-01

Graphene oxide nanosheets (GONs)/carboxymethyl chitosan (CMC)/Fe3O4 magnetic composite microspheres (MCMs) were prepared by enclosing Fe3O4 particles with CMC and GONs in turn. The microstructures of GONs and GONs/CMC/Fe3O4 MCMs were characterized by FTIR, XRD, TEM, and SEM. The effects of GON content, pH value, and adsorption time on the adsorption capacity of the MCMs were investigated. The results show that the GONs/CMC/Fe3O4 MCMs have a greater specific surface area and a strong adsorption capacity for dye wastewater. Meanwhile, the adsorption mechanism was investigated, and the results accorded with the pseudo-second-order kinetic model and the Freundlich isotherm model. The search results indicate that GONs/CMC/Fe3O4 MCMs can be used to purify dye wastewater and has an important potential use in the practical purification of dye wastewater. PMID:28772419

Ultrasound-Assisted Extraction, Antioxidant and Anticancer Activities of the Polysaccharides from Rhynchosia minima Root.

PubMed

Jia, Xuejing; Zhang, Chao; Hu, Jie; He, Muxue; Bao, Jiaolin; Wang, Kai; Li, Peng; Chen, Meiwan; Wan, Jianbo; Su, Huanxing; Zhang, Qingwen; He, Chengwei

2015-11-23

Box-Behnken design (BBD), one of the most common response surface methodology (RSM) methods, was used to optimize the experimental conditions for ultrasound-assisted extraction of polysaccharides from Rhynchosia minima root (PRM). The antioxidant abilities and anticancer activity of purified polysaccharide fractions were also measured. The results showed that optimal extraction parameters were as follows: ultrasound exposure time, 21 min; ratio of water to material, 46 mL/g; ultrasound extraction temperature, 63 °C. Under these conditions, the maximum yield of PRM was 16.95%±0.07%. Furthermore, the main monosaccharides of purified fractions were Ara and Gal. PRM3 and PRM5 exhibited remarkable DPPH radical scavenging activities and reducing power in vitro. PRM3 showed strong inhibitory activities on the growth of MCF-7 cells in vitro. The above results indicate that polysaccharides from R. minima root have the potential to be developed as natural antioxidants and anticancer ingredients for the food and pharmaceutical industries.
Mathematical modeling of the whole expanded bed adsorption process to recover and purify chitosanases from the unclarified fermentation broth of Paenibacillus ehimensis.

PubMed

de Araújo Padilha, Carlos Eduardo; Fortunato Dantas, Paulo Victor; de Sousa, Francisco Canindé; de Santana Souza, Domingos Fabiano; de Oliveira, Jackson Araújo; de Macedo, Gorete Ribeiro; Dos Santos, Everaldo Silvino

2016-12-15

In this study, a general rate model was applied to the entire process of expanded bed adsorption chromatography (EBAC) for the chitosanases purification protocol from unclarified fermentation broth produced by Paenibacillus ehimensis using the anionic adsorbent Streamline ® DEAE. For the experiments performed using the expanded bed, a homemade column (2.6cm×30.0cm) was specially designed. The proposed model predicted the entire EBA process adequately, giving R 2 values higher than 0.85 and χ 2 as low as 0.351 for the elution step. Using the validated model, a 3 3 factorial design was used to investigate other non-tested conditions as input. It was observed that the superficial velocity during loading and washing steps, as well as the settled bed height, has a strong positive effect on the F objective function used to evaluate the production of the purified chitosanases. Copyright © 2016 Elsevier B.V. All rights reserved.
Metal chelate affinity precipitation of RNA and purification of plasmid DNA

NASA Technical Reports Server (NTRS)

Balan, Sindhu; Murphy, Jason; Galaev, Igor; Kumar, Ashok; Fox, George E.; Mattiasson, Bo; Willson, Richard C.

2003-01-01

The affinity of metal chelates for amino acids, such as histidine, is widely used in purifying proteins, most notably through six-histidine 'tails'. We have found that metal affinity interactions can also be applied to separation of single-stranded nucleic acids through interactions involving exposed purines. Here we describe a metal affinity precipitation method to resolve RNA from linear and plasmid DNA. A copper-charged copolymer of N-isopropyl acrylamide (NIPAM) and vinyl imidazole (VI) is used to purify plasmid from an alkaline lysate of E. coli. The NIPAM units confer reversible solubility on the copolymer while the imidazole chelates metal ions in a manner accessible to interaction with soluble ligands. RNA was separated from the plasmid by precipitation along with the polymer in the presence of 800 mM NaCl. Bound RNA could be recovered by elution with imidazole and separated from copolymer by a second precipitation step. RNA binding showed a strong dependence on temperature and on the type of buffer used.
Photocatalytic treatment of bioaerosols: impact of the reactor design.

PubMed

Josset, Sébastien; Taranto, Jérôme; Keller, Nicolas; Keller, Valérie; Lett, Marie-Claire

2010-04-01

Comparing the UV-A photocatalytic treatment of bioaerosols contaminated with different airborne microorganisms such as L. pneumophila bacteria, T2 bacteriophage viruses and B. atrophaeus bacterial spores, pointed out a decontamination sensitivity following the bacteria > virus > bacterial spore ranking order, differing from that obtained for liquid-phase or surface UV-A photocatalytic disinfection. First-principles CFD investigation applied to a model annular photoreactor evidenced that larger the microorganism size, higher the hit probability with the photocatalytic surfaces. Applied to a commercial photocatalytic purifier case-study, the CFD calculations showed that the performances of the studied purifier could strongly benefit from rational reactor design engineering. The results obtained highlighted the required necessity to specifically investigate the removal of airborne microorganisms in terms of reactor design, and not to simply transpose the results obtained from studies performed toward chemical pollutants, especially for a successful commercial implementation of air decontamination photoreactors. This illustrated the importance of the aerodynamics in air decontamination, directly resulting from the microorganism morphology.
Recombinant DHX33 Protein Possesses Dual DNA/RNA Helicase Activity.

PubMed

Wang, Xingshun; Ge, Wei; Zhang, Yandong

2018-06-13

RNA helicase DHX33 has been shown to participate in a variety of cellular activities, including ribosome biogenesis, protein translation, and gene transcription. We and others further discovered that DHX33 is strongly expressed in several types of human cancers and plays important roles in promoting cancer cell proliferation. To better understand the molecular mechanism for DHX33 in exerting its biological functions, we purified recombinant DHX33 and performed biochemical studies in vitro. DHX33 protein was found to have ATPase activity that is dependent on DNA or RNA duplexes. The ATPase activity of DHX33 is coupled with its RNA/DNA unwinding activity. If a key residue in the ATP binding site were mutated, the mutant DHX33 could not unwind DNA/RNA duplexes. Furthermore, a deletion mutant of a RKK motif previously identified to be involved in ribosome DNA binding could still unwind DNA duplexes, albeit with reduced efficiency. In summary, our study reveals that purified DHX33 protein possesses unwinding activity toward DNA and RNA duplexes.
Electrophysiological characterization of the archaeal transporter NCX_Mj using solid supported membrane technology

PubMed Central

Barthmes, Maria; Liao, Jun; Jiang, Youxing; Brüggemann, Andrea

2016-01-01

Sodium–calcium exchangers (NCXs) are membrane transporters that play an important role in Ca2+ homeostasis and Ca2+ signaling. The recent crystal structure of NCX_Mj, a member of the NCX family from the archaebacterium Methanococcus jannaschii, provided insight into the atomistic details of sodium–calcium exchange. Here, we extend these findings by providing detailed functional data on purified NCX_Mj using solid supported membrane (SSM)–based electrophysiology, a powerful but unexploited tool for functional studies of electrogenic transporter proteins. We show that NCX_Mj is highly selective for Na+, whereas Ca2+ can be replaced by Mg2+ and Sr2+ and that NCX_Mj can be inhibited by divalent ions, particularly Cd2+. By directly comparing the apparent affinities of Na+ and Ca2+ for NCX_Mj with those for human NCX1, we show excellent agreement, indicating a strong functional similarity between NCX_Mj and its eukaryotic isoforms. We also provide detailed instructions to facilitate the adaption of this method to other electrogenic transporter proteins. Our findings demonstrate that NCX_Mj can serve as a model for the NCX family and highlight several possible applications for SSM-based electrophysiology. PMID:27241699
Evolutionary Patterns of RNA-Based Duplication in Non-Mammalian Chordates

PubMed Central

Li, Xin; Vibranovski, Maria D.; Gan, Xiaoni; Wang, Dengqiang; Wang, Wen; Long, Manyuan; He, Shunping

2011-01-01

The role of RNA-based duplication, or retroposition, in the evolution of new gene functions in mammals, plants, and Drosophila has been widely reported. However, little is known about RNA-based duplication in non-mammalian chordates. In this study, we screened ten non-mammalian chordate genomes for retrocopies and investigated their evolutionary patterns. We identified numerous retrocopies in these species. Examination of the age distribution of these retrocopies revealed no burst of young retrocopies in ancient chordate species. Upon comparing these non-mammalian chordate species to the mammalian species, we observed that a larger fraction of the non-mammalian retrocopies was under strong evolutionary constraints than mammalian retrocopies are, as evidenced by signals of purifying selection and expression profiles. For the Western clawed frog, Medaka, and Sea squirt, many retrogenes have evolved gonad and brain expression patterns, similar to what was observed in human. Testing of retrogene movement in the Medaka genome, where the nascent sex chrosomes have been well assembled, did not reveal any significant gene movement. Taken together, our analyses demonstrate that RNA-based duplication generates many functional genes and can make a significant contribution to the evolution of non-mammalian genomes. PMID:21779328
Enzymatic Properties of an Alkaline and Chelator Resistant alpha-amylase from the Alkaliphilic Bacillus sp. Isolate L1711

NASA Technical Reports Server (NTRS)

Bernhardsdotter, Eva C. M. J.; Pusey, Marc L.; Ng, Joseph D.; Garriott, Owen K.

2004-01-01

An alkaliphilic amylase producing bacterium, Bacillus sp. strain L 711, was selected among 13 soda lakes isolates. When grown at pH 10.5 and 37 C, strain L711 produced multiple forms of amylases in the culture broth. One of these, BAA, was purified from the culture supernatant by QAE column chromatography and preparative native gel electrophoresis. The molecular weight of BAA was determined to be 51 kDa by denaturing gel electrophoresis. The pH optima for activity below and above 40 C were 9.5 - 10.0 and 7.0 - 7.5 respectively. BAA was stable in the pH range 6-11 and was completely inactivated at 55 C. The thermostability was not increased in the presence of Ca(2+). The enzyme was strongly inhibited by Ca(2+), Zn(2+), Mg(2+), Mn(2+), Ba(2+) and Cu(2+), whereas the presence of Na(+), Co(2+) and EDTA (10 mM) enhanced enzymatic activity. The K(sub m), and specific activity of BAA on soluble starch were 1.9 mg/ml and 18.5 U/mg respectively. The main end products of hydrolysis were maltotetraose, maltose and glucose.
Enzymatic Properties of an Alkaline and Chelator Resistant Proportional to alpha-Amylase from the Alkaliphilic Bacillus sp. Isolate L1711

NASA Technical Reports Server (NTRS)

Bernhardsdotter, Eva C. M. J.; Pusey, Marc L.; Ng, Joseph D.; Garriott, Owen K.

2004-01-01

An alkaliphilic amylase producing bacterium, Bacillus sp. strain L1711, was selected among 13 soda lakes isolates. When grown at pH 10.5 and 370 C, strain L1711 produced multiple forms of amylases in the culture broth. One of these, BAA, was purified from the culture supernatant by QAE column chromatography and preparative native gel electrophoresis. The molecular weight of BAA was determined to be 51 kDa by denaturing gel electrophoresis. The pH optima for activity below and above 40 C were 9.5-10.0 and 7.0-7.5 respectively. BAA was stable in the pH range 6-11 and was completely inactivated at 55?C. The thermostability was not increased in the presence of Ca(2+). The enzyme was strongly inhibited by Ca(2+), Zn(2+), Mg(2+), Mn(2+), Ba(2+) and Cu(2+), whereas the presence of Na(+), Co2+ and EDTA (10 mM) enhanced enzymatic activity. The K(sub m) and specific activity of BAA on soluble starch were 1.9 mg/ml and 18.5 U/mg respectively. The main end products of hydrolysis were maltotetraose, maltose and glucose .
Lack of specific alleles for the bovine chemokine (C-X-C) receptor type 4 (CXCR4) gene in West African cattle questions its role as a candidate for trypanotolerance.

PubMed

Álvarez, Isabel; Pérez-Pardal, Lucía; Traoré, Amadou; Fernández, Iván; Goyache, Félix

2016-08-01

A panel of 81 Asian, African and European cattle (Bos taurus and B. indicus) was analysed for the whole sequence of the CXCR4 gene (3844bp), a strong candidate for cattle trypanotolerance. Thirty-one polymorphic sites identified gave 31 different haplotypes. Neutrality tests rejected the hypothesis of either positive or purifying selection. Bayesian phylogenetic tree showed differentiation of haplotypes into two clades gathering genetic variability predating domestication. Related with clades definition, linkage disequilibrium analyses suggested the existence of one only linkage block on the CXCR4 gene. Two tag SNPs identified on exon 2 captured 50% of variability. Whatever the analysis carried out, no clear separation between cattle groups was identified. Most haplotypes identified in West African taurine cattle were also found in European cattle and in Asian and West African zebu. West African taurine samples did not carry unique variants on the CXCR4 gene sequence. The current analysis failed in identifying a causal mutation on the CXCR4 gene underlying a previously reported QTL for cattle trypanotolerance on BTA2. Copyright © 2016 Elsevier B.V. All rights reserved.
Early-shared Mycobacterium bovis bacillus Calmette-Guérin sub-strains induce Th1 cytokine production in vivo.

PubMed

Taniguchi, Keiichi; Miyatake, Yuuji; Hayashi, Daisuke; Takami, Atsuro; Itoh, Saotomo; Yamamoto, Saburo; Hida, Shigeaki; Onozaki, Kikuo; Takii, Takemasa

2015-11-01

Interleukin-12 is one of the cytokines that induce acquired immunity by progressing the differentiation of T cells. When antigens are presented by APCs, including macrophages and DCs, T cells are activated and produce the Th1 cytokines IL-2 and IFN-γ. We have previously reported greater IL-12 production from macrophages infected with early-shared BCG sub-strains (ex. BCG-Japan, -Sweden) than from those infected with late-shared BCG (ex. BCG-Pasteur and -Connaught) . In this study, we investigated the Th1 cytokine-inducing activity of splenocytes co-cultured with BCG-infected DCs. Early-shared BCG-infected DCs produced IL-12 and TNF-α⋅ Furthermore, when they were co-cultured with purified protein derivative-stimulated DCs, the splenocytes of mice immunized with BCG-Tokyo/Japan produced more Th1 cytokine than did those of mice immunized with BCG-Connaught. In conclusion, early-shared BCG sub-strains more strongly induce Th1 cytokine production in vivo. This study provides basic information to inform the selection of candidates for primary vaccination. © 2015 The Societies and Wiley Publishing Asia Pty Ltd.
The evolution of genes encoding for green fluorescent proteins: insights from cephalochordates (amphioxus)

NASA Astrophysics Data System (ADS)

Yue, Jia-Xing; Holland, Nicholas D.; Holland, Linda Z.; Deheyn, Dimitri D.

2016-06-01

Green Fluorescent Protein (GFP) was originally found in cnidarians, and later in copepods and cephalochordates (amphioxus) (Branchiostoma spp). Here, we looked for GFP-encoding genes in Asymmetron, an early-diverged cephalochordate lineage, and found two such genes closely related to some of the Branchiostoma GFPs. Dim fluorescence was found throughout the body in adults of Asymmetron lucayanum, and, as in Branchiostoma floridae, was especially intense in the ripe ovaries. Spectra of the fluorescence were similar between Asymmetron and Branchiostoma. Lineage-specific expansion of GFP-encoding genes in the genus Branchiostoma was observed, largely driven by tandem duplications. Despite such expansion, purifying selection has strongly shaped the evolution of GFP-encoding genes in cephalochordates, with apparent relaxation for highly duplicated clades. All cephalochordate GFP-encoding genes are quite different from those of copepods and cnidarians. Thus, the ancestral cephalochordates probably had GFP, but since GFP appears to be lacking in more early-diverged deuterostomes (echinoderms, hemichordates), it is uncertain whether the ancestral cephalochordates (i.e. the common ancestor of Asymmetron and Branchiostoma) acquired GFP by horizontal gene transfer (HGT) from copepods or cnidarians or inherited it from the common ancestor of copepods and deuterostomes, i.e. the ancestral bilaterians.
Optimized labeling of membrane proteins for applications to super-resolution imaging in confined cellular environments using monomeric streptavidin.

PubMed

Chamma, Ingrid; Rossier, Olivier; Giannone, Grégory; Thoumine, Olivier; Sainlos, Matthieu

2017-04-01

Recent progress in super-resolution imaging (SRI) has created a strong need to improve protein labeling with probes of small size that minimize the target-to-label distance, increase labeling density, and efficiently penetrate thick biological tissues. This protocol describes a method for labeling genetically modified proteins incorporating a small biotin acceptor peptide with a 3-nm fluorescent probe, monomeric streptavidin. We show how to express, purify, and conjugate the probe to organic dyes with different fluorescent properties, and how to label selectively biotinylated membrane proteins for SRI techniques (point accumulation in nanoscale topography (PAINT), stimulated emission depletion (STED), stochastic optical reconstruction microscopy (STORM)). This method is complementary to the previously described anti-GFP-nanobody/SNAP-tag strategies, with the main advantage being that it requires only a short 15-amino-acid tag, and can thus be used with proteins resistant to fusion with large tags and for multicolor imaging. The protocol requires standard molecular biology/biochemistry equipment, making it easily accessible for laboratories with only basic skills in cell biology and biochemistry. The production/purification/conjugation steps take ∼5 d, and labeling takes a few minutes to an hour.
Origins and Structural Properties of Novel and De Novo Protein Domains During Insect Evolution.

PubMed

Klasberg, Steffen; Bitard-Feildel, Tristan; Callebaut, Isabelle; Bornberg-Bauer, Erich

2018-05-26

Over long time scales, protein evolution is characterised by modular rearrangements of protein domains. Such rearrangements are mainly caused by gene duplication, fusion and terminal losses. To better understand domain emergence mechanisms we investigated 32 insect genomes covering a speciation gradient ranging from ~ 2 to ~ 390 my. We use established domain models and foldable domains delineated by Hydrophobic-Cluster-Analysis (HCA), which does not require homologous sequences, to also identify domains which have likely arisen de novo, i.e. from previously non-coding DNA. Our results indicate that most novel domains emerge terminally as they originate from ORF extensions while fewer arise in middle arrangements, resulting from exonisation of intronic or intergenic regions. Many novel domains rapidly migrate between terminal or middle positions and single- and multi-domain arrangements. Young domains, such as most HCA defined domains, are under strong selection pressure as they show signals of purifying selection. De novo domains, linked to ancient domains or defined by HCA, have higher degrees of intrinsic disorder and disorder-to-order transition upon binding than ancient domains. However, the corresponding DNA sequences of the novel domains of denovo origins could only rarely be found in sister genomes. We conclude that novel domains are often recruited by other proteins and undergo important structural modifications shortly after their emergence, but evolve too fast to be characterised by cross-species comparisons alone. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.
Purification of Immature Neuronal Cells from Neural Stem Cell Progeny

PubMed Central

Azari, Hassan; Osborne, Geoffrey W.; Yasuda, Takahiro; Golmohammadi, Mohammad G.; Rahman, Maryam; Deleyrolle, Loic P.; Esfandiari, Ebrahim; Adams, David J.; Scheffler, Bjorn; Steindler, Dennis A.; Reynolds, Brent A.

2011-01-01

Large-scale proliferation and multi-lineage differentiation capabilities make neural stem cells (NSCs) a promising renewable source of cells for therapeutic applications. However, the practical application for neuronal cell replacement is limited by heterogeneity of NSC progeny, relatively low yield of neurons, predominance of astrocytes, poor survival of donor cells following transplantation and the potential for uncontrolled proliferation of precursor cells. To address these impediments, we have developed a method for the generation of highly enriched immature neurons from murine NSC progeny. Adaptation of the standard differentiation procedure in concert with flow cytometry selection, using scattered light and positive fluorescent light selection based on cell surface antibody binding, provided a near pure (97%) immature neuron population. Using the purified neurons, we screened a panel of growth factors and found that bone morphogenetic protein-4 (BMP-4) demonstrated a strong survival effect on the cells in vitro, and enhanced their functional maturity. This effect was maintained following transplantation into the adult mouse striatum where we observed a 2-fold increase in the survival of the implanted cells and a 3-fold increase in NeuN expression. Additionally, based on the neural-colony forming cell assay (N-CFCA), we noted a 64 fold reduction of the bona fide NSC frequency in neuronal cell population and that implanted donor cells showed no signs of excessive or uncontrolled proliferation. The ability to provide defined neural cell populations from renewable sources such as NSC may find application for cell replacement therapies in the central nervous system. PMID:21687800
A phage display selected 7-mer peptide inhibitor of the Tannerella forsythia metalloprotease-like enzyme Karilysin can be truncated to Ser-Trp-Phe-Pro.

PubMed

Skottrup, Peter Durand; Sørensen, Grete; Ksiazek, Miroslaw; Potempa, Jan; Riise, Erik

2012-01-01

Tannerella forsythia is a gram-negative bacteria, which is strongly associated with the development of periodontal disease. Karilysin is a newly identified metalloprotease-like enzyme, that is secreted from T. forsythia. Karilysin modulates the host immune response and is therefore considered a likely drug target. In this study peptides were selected towards the catalytic domain from Karilysin (Kly18) by phage display. The peptides were linear with low micromolar binding affinities. The two best binders (peptide14 and peptide15), shared the consensus sequence XWFPXXXGGG. A peptide15 fusion with Maltose Binding protein (MBP) was produced with peptide15 fused to the N-terminus of MBP. The peptide15-MBP was expressed in E. coli and the purified fusion-protein was used to verify Kly18 specific binding. Chemically synthesised peptide15 (SWFPLRSGGG) could inhibit the enzymatic activity of both Kly18 and intact Karilysin (Kly48). Furthermore, peptide15 could slow down the autoprocessing of intact Kly48 to Kly18. The WFP motif was important for inhibition and a truncation study further demonstrated that the N-terminal serine was also essential for Kly18 inhibition. The SWFP peptide had a Ki value in the low micromolar range, which was similar to the intact peptide15. In conclusion SWFP is the first reported inhibitor of Karilysin and can be used as a valuable tool in structure-function studies of Karilysin.
Population Genomics of Daphnia pulex

PubMed Central

Lynch, Michael; Gutenkunst, Ryan; Ackerman, Matthew; Spitze, Ken; Ye, Zhiqiang; Maruki, Takahiro; Jia, Zhiyuan

2017-01-01

Using data from 83 isolates from a single population, the population genomics of the microcrustacean Daphnia pulex are described and compared to current knowledge for the only other well-studied invertebrate, Drosophila melanogaster. These two species are quite similar with respect to effective population sizes and mutation rates, although some features of recombination appear to be different, with linkage disequilibrium being elevated at short (<100 bp) distances in D. melanogaster and at long distances in D. pulex. The study population adheres closely to the expectations under Hardy–Weinberg equilibrium, and reflects a past population history of no more than a twofold range of variation in effective population size. Fourfold redundant silent sites and a restricted region of intronic sites appear to evolve in a nearly neutral fashion, providing a powerful tool for population genetic analyses. Amino acid replacement sites are predominantly under strong purifying selection, as are a large fraction of sites in UTRs and intergenic regions, but the majority of SNPs at such sites that rise to frequencies >0.05 appear to evolve in a nearly neutral fashion. All forms of genomic sites (including replacement sites within codons, and intergenic and UTR regions) appear to be experiencing an ∼2× higher level of selection scaled to the power of drift in D. melanogaster, but this may in part be a consequence of recent demographic changes. These results establish D. pulex as an excellent system for future work on the evolutionary genomics of natural populations. PMID:27932545
Large-Scale Phylogenomic Analysis Reveals the Complex Evolutionary History of Rabies Virus in Multiple Carnivore Hosts

PubMed Central

Troupin, Cécile; Dacheux, Laurent; Tanguy, Marion; Sabeta, Claude; Blanc, Hervé; Bouchier, Christiane; Vignuzzi, Marco; Holmes, Edward C.; Bourhy, Hervé

2016-01-01

The natural evolution of rabies virus (RABV) provides a potent example of multiple host shifts and an important opportunity to determine the mechanisms that underpin viral emergence. Using 321 genome sequences spanning an unprecedented diversity of RABV, we compared evolutionary rates and selection pressures in viruses sampled from multiple primary host shifts that occurred on various continents. Two major phylogenetic groups, bat-related RABV and dog-related RABV, experiencing markedly different evolutionary dynamics were identified. While no correlation between time and genetic divergence was found in bat-related RABV, the evolution of dog-related RABV followed a generally clock-like structure, although with a relatively low evolutionary rate. Subsequent molecular clock dating indicated that dog-related RABV likely underwent a rapid global spread following the intensification of intercontinental trade starting in the 15th century. Strikingly, although dog RABV has jumped to various wildlife species from the order Carnivora, we found no clear evidence that these host-jumping events involved adaptive evolution, with RABV instead characterized by strong purifying selection, suggesting that ecological processes also play an important role in shaping patterns of emergence. However, specific amino acid changes were associated with the parallel emergence of RABV in ferret-badgers in Asia, and some host shifts were associated with increases in evolutionary rate, particularly in the ferret-badger and mongoose, implying that changes in host species can have important impacts on evolutionary dynamics. PMID:27977811
Large-Scale Phylogenomic Analysis Reveals the Complex Evolutionary History of Rabies Virus in Multiple Carnivore Hosts.

PubMed

Troupin, Cécile; Dacheux, Laurent; Tanguy, Marion; Sabeta, Claude; Blanc, Hervé; Bouchier, Christiane; Vignuzzi, Marco; Duchene, Sebastián; Holmes, Edward C; Bourhy, Hervé

2016-12-01

The natural evolution of rabies virus (RABV) provides a potent example of multiple host shifts and an important opportunity to determine the mechanisms that underpin viral emergence. Using 321 genome sequences spanning an unprecedented diversity of RABV, we compared evolutionary rates and selection pressures in viruses sampled from multiple primary host shifts that occurred on various continents. Two major phylogenetic groups, bat-related RABV and dog-related RABV, experiencing markedly different evolutionary dynamics were identified. While no correlation between time and genetic divergence was found in bat-related RABV, the evolution of dog-related RABV followed a generally clock-like structure, although with a relatively low evolutionary rate. Subsequent molecular clock dating indicated that dog-related RABV likely underwent a rapid global spread following the intensification of intercontinental trade starting in the 15th century. Strikingly, although dog RABV has jumped to various wildlife species from the order Carnivora, we found no clear evidence that these host-jumping events involved adaptive evolution, with RABV instead characterized by strong purifying selection, suggesting that ecological processes also play an important role in shaping patterns of emergence. However, specific amino acid changes were associated with the parallel emergence of RABV in ferret-badgers in Asia, and some host shifts were associated with increases in evolutionary rate, particularly in the ferret-badger and mongoose, implying that changes in host species can have important impacts on evolutionary dynamics.
Allium sativum Protease Inhibitor: A Novel Kunitz Trypsin Inhibitor from Garlic Is a New Comrade of the Serpin Family.

PubMed

Shamsi, Tooba Naz; Parveen, Romana; Amir, Mohd; Baig, Mohd Affan; Qureshi, M Irfan; Ali, Sher; Fatima, Sadaf

2016-01-01

This study was aimed to purify and characterize the Protease inhibitor (PI) from a plant Allium sativum (garlic) with strong medicinal properties and to explore its phytodrug potentials. Allium sativum Protease Inhibitor (ASPI) was purified using ammonium sulphate fractionation and Fast Protein Liquid Chromatography on anion exchanger Hi-Trap DEAE column. The purified protein was analyzed for its purity and molecular weight by SDS PAGE. The confirmation of presence of trypsin inhibiting PI was performed by MALDI TOF-TOF and analyzed by MASCOT database. The ASPI was further investigated for its kinetic properties and stability under extreme conditions of pH, temperature and chemical denaturants. Secondary structure was determined by Circular Dichorism (CD) spectroscopy. ASPI of ~15 kDa inhibited trypsin and matched "truncated kunitz Trypsin Inhibitor (Glycine max)" in MASCOT database. The purified ASPI showed 30376.1371 U/mg specific activity with a fold purity of 159.92 and yield ~93%. ASPI was quite stable in the range of pH 2-12 showing a decline in the activity around pH 4-5 suggesting that the pI value of the protein as ASPI aggregates in this range. ASPI showed stability to a broad range of temperature (10-80°C) but declined beyond 80°C. Further, detergents, oxidizing agents and reducing agents demonstrated change in ASPI activity under varying concentrations. The kinetic analysis revealed sigmoidal relationship of velocity with substrate concentration with Vmax 240.8 (μM/min) and Km value of 0.12 μM. ASPI showed uncompetitive inhibition with a Ki of 0.08±0.01 nM). The Far UV CD depicted 2.0% α -helices and 51% β -sheets at native pH. To conclude, purified ~15 kDa ASPI exhibited fair stability in wide range of pH and temperature Overall, there was an increase in purification fold with remarkable yield. Chemical modification studies suggested the presence of lysine and tryptophan residues as lead amino acids present in the reactive sites. Therefore, ASPI with trypsin inhibitory property has the potential to be used as a non-cytotoxic clinical agents.

Adsorption of benzene, toluene, and xylene by two tetramethylammonium-smectites having different charge densities

USGS Publications Warehouse

Lee, Jiunn-Fwu; Mortland, Max M.; Chiou, Cary T.; Kite, Daniel E.; Boyd, Stephen A.

1990-01-01

A high-charge smectite from Arizona [cation-exchange capacity (CEC) = 120 meq/100 g] and a low-charge smectite from Wyoming (CEC = 90 meq/100 g) were used to prepare homoionic tetramethylammonium (TMA)-clay complexes. The adsorption of benzene, toluene, and o-xylene as vapors by the dry TMA-clays and as solutes from water by the wet TMA-clays was studied. The adsorption of the organic vapors by the dry TMA-smectite samples was strong and apparently consisted of interactions with both the aluminosilicate mineral surfaces and the TMA exchange ions in the interlayers. In the adsorption of organic vapors, the closer packing of TMA ions in the dry high-charge TMA-smectite, compared with the dry low-charge TMA-smectite, resulted in a somewhat higher degree of shape-selective adsorption of benzene, toluene, and xylene. In the presence of water, the adsorption capacities of both samples for the aromatic compounds were significantly reduced, although the uptake of benzene from water by the low-charge TMA-smectite was still substantial. This lower sorption capacity was accompanied by increased shape-selectivity for the aromatic compounds. The reduction in uptake and increased selectivity was much more pronounced for the water-saturated, high-charge TMA-smectite than for the low-charge TMA-smectite. Hydration of the TMA exchange ions and/or the mineral surfaces apparently reduced the accessibility of the aromatic molecules to interlamellar regions. The resulting water-induced sieving effect was greater for the high-charge TMA-smectite due to the higher density of exchanged TMA-ions. The low-charge Wyoming TMA-smectite was a highly effective adsorbent for removing benzene from water and may be useful for purifying benzene-contaminated water.
Theory of microbial genome evolution

NASA Astrophysics Data System (ADS)

Koonin, Eugene

Bacteria and archaea have small genomes tightly packed with protein-coding genes. This compactness is commonly perceived as evidence of adaptive genome streamlining caused by strong purifying selection in large microbial populations. In such populations, even the small cost incurred by nonfunctional DNA because of extra energy and time expenditure is thought to be sufficient for this extra genetic material to be eliminated by selection. However, contrary to the predictions of this model, there exists a consistent, positive correlation between the strength of selection at the protein sequence level, measured as the ratio of nonsynonymous to synonymous substitution rates, and microbial genome size. By fitting the genome size distributions in multiple groups of prokaryotes to predictions of mathematical models of population evolution, we show that only models in which acquisition of additional genes is, on average, slightly beneficial yield a good fit to genomic data. Thus, the number of genes in prokaryotic genomes seems to reflect the equilibrium between the benefit of additional genes that diminishes as the genome grows and deletion bias. New genes acquired by microbial genomes, on average, appear to be adaptive. Evolution of bacterial and archaeal genomes involves extensive horizontal gene transfer and gene loss. Many microbes have open pangenomes, where each newly sequenced genome contains more than 10% `ORFans', genes without detectable homologues in other species. A simple, steady-state evolutionary model reveals two sharply distinct classes of microbial genes, one of which (ORFans) is characterized by effectively instantaneous gene replacement, whereas the other consists of genes with finite, distributed replacement rates. These findings imply a conservative estimate of at least a billion distinct genes in the prokaryotic genomic universe.
Genetic analyses of bolting in bulb onion (Allium cepa L.).

PubMed

Baldwin, Samantha; Revanna, Roopashree; Pither-Joyce, Meeghan; Shaw, Martin; Wright, Kathryn; Thomson, Susan; Moya, Leire; Lee, Robyn; Macknight, Richard; McCallum, John

2014-03-01

We present the first evidence for a QTL conditioning an adaptive trait in bulb onion, and the first linkage and population genetics analyses of candidate genes involved in photoperiod and vernalization physiology. Economic production of bulb onion (Allium cepa L.) requires adaptation to photoperiod and temperature such that a bulb is formed in the first year and a flowering umbel in the second. 'Bolting', or premature flowering before bulb maturation, is an undesirable trait strongly selected against by breeders during adaptation of germplasm. To identify genome regions associated with adaptive traits we conducted linkage mapping and population genetic analyses of candidate genes, and QTL analysis of bolting using a low-density linkage map. We performed tagged amplicon sequencing of ten candidate genes, including the FT-like gene family, in eight diverse populations to identify polymorphisms and seek evidence of differentiation. Low nucleotide diversity and negative estimates of Tajima's D were observed for most genes, consistent with purifying selection. Significant population differentiation was observed only in AcFT2 and AcSOC1. Selective genotyping in a large 'Nasik Red × CUDH2150' F2 family revealed genome regions on chromosomes 1, 3 and 6 associated (LOD > 3) with bolting. Validation genotyping of two F2 families grown in two environments confirmed that a QTL on chromosome 1, which we designate AcBlt1, consistently conditions bolting susceptibility in this cross. The chromosome 3 region, which coincides with a functionally characterised acid invertase, was not associated with bolting in other environments, but showed significant association with bulb sucrose content in this and other mapping pedigrees. These putative QTL and candidate genes were placed on the onion map, enabling future comparative studies of adaptive traits.
Selection and reduced population size cannot explain higher amounts of Neandertal ancestry in East Asian than in European human populations.

PubMed

Kim, Bernard Y; Lohmueller, Kirk E

2015-03-05

It has been hypothesized that the greater proportion of Neandertal ancestry in East Asians than in Europeans is due to the fact that purifying selection is less effective at removing weakly deleterious Neandertal alleles from East Asian populations. Using simulations of a broad range of models of selection and demography, we have shown that this hypothesis cannot account for the higher proportion of Neandertal ancestry in East Asians than in Europeans. Instead, more complex demographic scenarios, most likely involving multiple pulses of Neandertal admixture, are required to explain the data. Copyright © 2015 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.
Single cell gene expression profiling of cortical osteoblast lineage cells.

PubMed

Flynn, James M; Spusta, Steven C; Rosen, Clifford J; Melov, Simon

2013-03-01

In tissues with complex architectures such as bone, it is often difficult to purify and characterize specific cell types via molecular profiling. Single cell gene expression profiling is an emerging technology useful for characterizing transcriptional profiles of individual cells isolated from heterogeneous populations. In this study we describe a novel procedure for the isolation and characterization of gene expression profiles of single osteoblast lineage cells derived from cortical bone. Mixed populations of different cell types were isolated from adult long bones of C57BL/6J mice by enzymatic digestion, and subsequently subjected to FACS to purify and characterize osteoblast lineage cells via a selection strategy using antibodies against CD31, CD45, and alkaline phosphatase (AP), specific for mature osteoblasts. The purified individual osteoblast lineage cells were then profiled at the single cell level via nanofluidic PCR. This method permits robust gene expression profiling on single osteoblast lineage cells derived from mature bone, potentially from anatomically distinct sites. In conjunction with this technique, we have also shown that it is possible to carry out single cell profiling on cells purified from fixed and frozen bone samples without compromising the gene expression signal. The latter finding means the technique can be extended to biopsies of bone from diseased individuals. Our approach for single cell expression profiling provides a new dimension to the transcriptional profile of the primary osteoblast lineage population in vivo, and has the capacity to greatly expand our understanding of how these cells may function in vivo under normal and diseased states. Copyright © 2012 Elsevier Inc. All rights reserved.
Purification Or Organic Acids Using Anion Exchange Chromatography.

DOEpatents

Ponnampalam; Elankovan

2001-09-04

Disclosed is a cost-effective method for purifying and acidifying carboxylic acids, including organic acids and amino acids. The method involves removing impurities by allowing the anionic form of the carboxylic acid to bind to an anion exchange column and washing the column. The carboxylic anion is displaced as carboxylic acid by washing the resin with a strong inorganic anion. This method is effective in removing organic carboxylic acids and amino acids from a variety of industrial sources, including fermentation broths, hydrolysates, and waste streams.
Purification, physicochemical characterization, saccharide specificity, and chemical modification of a Gal/GalNAc specific lectin from the seeds of Trichosanthes dioica.

PubMed

Sultan, Nabil Ali Mohammed; Kenoth, Roopa; Swamy, Musti J

2004-12-15

A new galactose-specific lectin has been purified from the extracts of Trichosanthes dioica seeds by affinity chromatography on cross-linked guar gum. The purified lectin (T. dioica seed lectin, TDSL) moved as a single symmetrical peak on gel filtration on Superose-12 in the presence of 0.1 M lactose with an M(r) of 55 kDa. In the absence of ligand, the movement was retarded, indicating a possible interaction of the lectin with the column matrix. In SDS-PAGE, in the presence of beta-mercaptoethanol, two non-identical bands of M(r) 24 and 37 kDa were observed, whereas in the absence of beta-mercaptoethanol, the lectin yielded a single band corresponding to approximately 55,000 Da, indicating that the two subunits of TDSL are connected by one or more disulfide bridges. TDSL is a glycoprotein with about 4.9% covalently bound neutral sugar. Analysis of near-UV CD spectrum by three different methods (CDSSTR, CONTINLL, and SELCON3) shows that TDSL contains 13.3% alpha-helix, 36.7% beta-sheet, 19.4% beta-turns, and 31.6% unordered structure. Among a battery of sugars investigated, TDSL was inhibited strongly by beta-d-galactopyranosides, with 4-methylumbelliferyl-beta-d-galactopyranoside being the best ligand. Chemical modification studies indicate that tyrosine residues are important for the carbohydrate-binding and hemagglutinating activities of the lectin. A partial protection was observed when the tyrosine modification was performed in the presence of 0.2 M lactose. The tryptophan residues of TDSL appear to be buried in the protein interior as they could not be modified under native conditions, whereas upon denaturation with 8 M urea two Trp residues could be selectively modified by N-bromosuccinimide. The subunit composition and size, secondary structure, and sugar specificity of this lectin are similar to those of type-2 ribosome inactivating proteins, suggesting that TDSL may belong to this protein family.
Isolation and Structural Elucidation of Brevibacillin, an Antimicrobial Lipopeptide from Brevibacillus laterosporus That Combats Drug-Resistant Gram-Positive Bacteria.

PubMed

Yang, Xu; Huang, En; Yuan, Chunhua; Zhang, Liwen; Yousef, Ahmed E

2016-05-01

A new environmental bacterial strain exhibited strong antimicrobial characteristics against methicillin-resistant Staphylococcus aureus, vancomycin-resistant strains of Enterococcus faecalis and Lactobacillus plantarum, and other Gram-positive bacteria. The producer strain, designated OSY-I1, was determined to be Brevibacillus laterosporusvia morphological, biochemical, and genetic analyses. The antimicrobial agent was extracted from cells of OSY-I1 with isopropanol, purified by high-performance liquid chromatography, and structurally analyzed using mass spectrometry (MS) and nuclear magnetic resonance (NMR). The MS and NMR results, taken together, uncovered a linear lipopeptide consisting of 13 amino acids and an N-terminal C6 fatty acid (FA) chain, 2-hydroxy-3-methylpentanoic acid. The lipopeptide (FA-Dhb-Leu-Orn-Ile-Ile-Val-Lys-Val-Val-Lys-Tyr-Leu-valinol, where Dhb is α,β-didehydrobutyric acid and valinol is 2-amino-3-methyl-1-butanol) has a molecular mass of 1,583.0794 Da and contains three modified amino acid residues: α,β-didehydrobutyric acid, ornithine, and valinol. The compound, designated brevibacillin, was determined to be a member of a cationic lipopeptide antibiotic family. In addition to its potency against drug-resistant bacteria, brevibacillin also exhibited low MICs (1 to 8 μg/ml) against selected foodborne pathogenic and spoilage bacteria, such as Listeria monocytogenes,Bacillus cereus, and Alicyclobacillus acidoterrestris Purified brevibacillin showed no sign of degradation when it was held at 80 °C for 60 min, and it retained at least 50% of its antimicrobial activity when it was held for 22 h under acidic or alkaline conditions. On the basis of these findings, brevibacillin is a potent antimicrobial lipopeptide which is potentially useful to combat drug-resistant bacterial pathogens and foodborne pathogenic and spoilage bacteria. Copyright © 2016, American Society for Microbiology. All Rights Reserved.
High-throughput process development of an alternative platform for the production of virus-like particles in Escherichia coli.

PubMed

Ladd Effio, Christopher; Baumann, Pascal; Weigel, Claudia; Vormittag, Philipp; Middelberg, Anton; Hubbuch, Jürgen

2016-02-10

The production of safe vaccines against untreatable or new diseases has pushed the research in the field of virus-like particles (VLPs). Currently, a large number of commercial VLP-based human vaccines and vaccine candidates are available or under development. A promising VLP production route is the controlled in vitro assembly of virus proteins into capsids. In the study reported here, a high-throughput screening (HTS) procedure was implemented for the upstream process development of a VLP platform in bacterial cell systems. Miniaturized cultivations were carried out in 48-well format in the BioLector system (m2p-Labs, Germany) using an Escherichia coli strain with a tac promoter producing the murine polyomavirus capsid protein (VP1). The screening procedure incorporated micro-scale cultivations, HTS cell disruption by sonication and HTS-compatible analytics by capillary gel electrophoresis. Cultivation temperatures, shaking speeds, induction and medium conditions were varied to optimize the product expression in E. coli. The most efficient system was selected based on an evaluation of soluble and insoluble product concentrations as well as on the percentage of product in the total soluble protein fraction. The optimized system was scaled up to cultivation 2.5L shaker flask scale and purified using an anion exchange chromatography membrane adsorber, followed by a size exclusion chromatography polishing procedure. For proof of concept, purified VP1 capsomeres were assembled under defined buffer conditions into empty capsids and characterized using transmission electron microscopy (TEM). The presented HTS procedure allowed for a fast development of an efficient production process of VLPs in E. coli. Under optimized cultivation conditions, the VP1 product totalled up to 43% of the total soluble protein fraction, yielding 1.63 mg VP1 per mL of applied cultivation medium. The developed production process strongly promotes the murine polyoma-VLP platform, moving towards an industrially feasible technology for new chimeric vaccines. Copyright © 2015 Elsevier B.V. All rights reserved.
Immunological and biochemical analysis of glycosylated surface antigens and lipophosphoglycan of Tritrichomonas foetus.

PubMed

Singh, B N; BonDurant, R H; Campero, C M; Corbeil, L B

2001-08-01

Immunoaffinity-purified TF1.17 adhesin antigen was compared biochemically and antigenically to Tritrichomonas foetus (TF) lipophosphoglycan (LPG) and a soluble glycosylated antigen (SGA) released from T. foetus and implicated in pathogenesis and immunity. The monoclonal antibodies (Mabs TF1.15 and TF1.17) specific for a glycosylated TF1.17 antigen were previously shown to prevent adhesion of the T. foetus parasites to bovine vaginal epithelial cells and to mediate killing by bovine complement. SGA was isolated from T. foetus-conditioned buffer and purified by octyl-Sepharose hydrophobic column chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of SGA showed a major SGA1 component (approximately 190 kDa) and a minor SGA2 component (50-70 kDa), which migrated close to TF-LPG and TF1.17. The carbohydrate and lipid compositional analyses of affinity-purified TF1.17 and SGA2 by high-performance liquid chromatography (HPLC) and gas-liquid chromatography revealed the presence of monosaccharides and fatty acids as found in TF-LPG. All antigens contained terminal fucose as determined by alpha-fucosidase digestion followed by HPLC. ELISA and western blots were used to further characterize these glycosylated antigens and to analyze their relationships. The Mabs TF1.15 and TF1.17 reacted very strongly to TF-LPG and SGA2. as well as TF1.17 antigen, indicating that these molecules share common epitopes. These Mabs did not react with the SGA1 component either in ELISA and western blot analyses. Also, the monosaccharide composition of SGA1 was very different from the other three antigen, suggesting SGA1 was different from LPG, SGA2 and TF1.17. Although LPG reacted with Mabs to native TF1.17 antigen, LPG did not induce an immune response in cattle with the same route and adjuvant used to produce strong antibody responses to the native antigen. The latter response suggests that the tightly bound peptide present in the immunoaffinity-purified antigen is necessary for induction of a response to (an) epitope(s) in TF-LPG and TF1.17. Furthermore, vaginal fluid from T. foetus-infected heifers and serum from a cow with a T. foetus-associated pyometra recognized both TF1.17 and TF-LPG in western blots. These results suggest that T. foetus LPG and SGA2 are related to TF1.17 antigen, which was previously shown to play an important role in the pathogenesis and host response in bovine trichomoniasis.
Comparative study on the selective chalcopyrite bioleaching of a molybdenite concentrate with mesophilic and thermophilic bacteria.

PubMed

Romano, P; Blázquez, M L; Alguacil, F J; Muñoz, J A; Ballester, A; González, F

2001-03-01

This study evaluates different bioleaching treatments of a molybdenite concentrate using mesophilic and thermophilic bacterial cultures. Further studies on the chemical leaching and the electrochemical behavior of the MoS(2) concentrate were carried out. Bioleaching tests showed a progressive removal of chalcopyrite from the molybdenite concentrate with an increase in temperature. Chemical leaching tests support the idea of an indirect attack of the concentrate. Electrochemical tests indicate that chalcopyrite dissolution is favored when molybdenite is present. Therefore, this type of bioleaching treatment could be applied to purify molybdenite flotation concentrates by selectively dissolving chalcopyrite.
Whole-Gene Positive Selection, Elevated Synonymous Substitution Rates, Duplication, and Indel Evolution of the Chloroplast clpP1 Gene

PubMed Central

Erixon, Per; Oxelman, Bengt

2008-01-01

Background Synonymous DNA substitution rates in the plant chloroplast genome are generally relatively slow and lineage dependent. Non-synonymous rates are usually even slower due to purifying selection acting on the genes. Positive selection is expected to speed up non-synonymous substitution rates, whereas synonymous rates are expected to be unaffected. Until recently, positive selection has seldom been observed in chloroplast genes, and large-scale structural rearrangements leading to gene duplications are hitherto supposed to be rare. Methodology/Principle Findings We found high substitution rates in the exons of the plastid clpP1 gene in Oenothera (the Evening Primrose family) and three separate lineages in the tribe Sileneae (Caryophyllaceae, the Carnation family). Introns have been lost in some of the lineages, but where present, the intron sequences have substitution rates similar to those found in other introns of their genomes. The elevated substitution rates of clpP1 are associated with statistically significant whole-gene positive selection in three branches of the phylogeny. In two of the lineages we found multiple copies of the gene. Neighboring genes present in the duplicated fragments do not show signs of elevated substitution rates or positive selection. Although non-synonymous substitutions account for most of the increase in substitution rates, synonymous rates are also markedly elevated in some lineages. Whereas plant clpP1 genes experiencing negative (purifying) selection are characterized by having very conserved lengths, genes under positive selection often have large insertions of more or less repetitive amino acid sequence motifs. Conclusions/Significance We found positive selection of the clpP1 gene in various plant lineages to correlated with repeated duplication of the clpP1 gene and surrounding regions, repetitive amino acid sequences, and increase in synonymous substitution rates. The present study sheds light on the controversial issue of whether negative or positive selection is to be expected after gene duplications by providing evidence for the latter alternative. The observed increase in synonymous substitution rates in some of the lineages indicates that the detection of positive selection may be obscured under such circumstances. Future studies are required to explore the functional significance of the large inserted repeated amino acid motifs, as well as the possibility that synonymous substitution rates may be affected by positive selection. PMID:18167545
Production of capsular polysaccharide of Streptococcus pneumoniae type 14 and its purification by affinity chromatography.

PubMed

Suárez, N; Fraguas, L F; Texeira, E; Massaldi, H; Batista-Viera, F; Ferreira, F

2001-02-01

We describe a rapid and efficient method for producing the capsular polysaccharide of Streptococcus pneumoniae by fermentation on tryptic soy broth and purification of this compound by using immobilized soybean lectin as an affinity adsorbent. In principle, the same strategy can be used to produce purified capsular polysaccharides from other streptococcal serotypes by selecting the appropriate lectin adsorbents.
Production of Capsular Polysaccharide of Streptococcus pneumoniae Type 14 and Its Purification by Affinity Chromatography

PubMed Central

Suárez, Norma; Fraguas, Laura Franco; Texeira, Esther; Massaldi, Hugo; Batista-Viera, Francisco; Ferreira, Fernando

2001-01-01

We describe a rapid and efficient method for producing the capsular polysaccharide of Streptococcus pneumoniae by fermentation on tryptic soy broth and purification of this compound by using immobilized soybean lectin as an affinity adsorbent. In principle, the same strategy can be used to produce purified capsular polysaccharides from other streptococcal serotypes by selecting the appropriate lectin adsorbents. PMID:11157270
Evolutionary games on cycles with strong selection

NASA Astrophysics Data System (ADS)

Altrock, P. M.; Traulsen, A.; Nowak, M. A.

2017-02-01

Evolutionary games on graphs describe how strategic interactions and population structure determine evolutionary success, quantified by the probability that a single mutant takes over a population. Graph structures, compared to the well-mixed case, can act as amplifiers or suppressors of selection by increasing or decreasing the fixation probability of a beneficial mutant. Properties of the associated mean fixation times can be more intricate, especially when selection is strong. The intuition is that fixation of a beneficial mutant happens fast in a dominance game, that fixation takes very long in a coexistence game, and that strong selection eliminates demographic noise. Here we show that these intuitions can be misleading in structured populations. We analyze mean fixation times on the cycle graph under strong frequency-dependent selection for two different microscopic evolutionary update rules (death-birth and birth-death). We establish exact analytical results for fixation times under strong selection and show that there are coexistence games in which fixation occurs in time polynomial in population size. Depending on the underlying game, we observe inherence of demographic noise even under strong selection if the process is driven by random death before selection for birth of an offspring (death-birth update). In contrast, if selection for an offspring occurs before random removal (birth-death update), then strong selection can remove demographic noise almost entirely.
Identification of conformational epitopes for human IgG on Chemotaxis inhibitory protein of Staphylococcus aureus

PubMed Central

Gustafsson, Erika; Haas, Pieter-Jan; Walse, Björn; Hijnen, Marcel; Furebring, Christina; Ohlin, Mats; van Strijp, Jos AG; van Kessel, Kok PM

2009-01-01

Background The Chemotaxis inhibitory protein of Staphylococcus aureus (CHIPS) blocks the Complement fragment C5a receptor (C5aR) and formylated peptide receptor (FPR) and is thereby a potent inhibitor of neutrophil chemotaxis and activation of inflammatory responses. The majority of the healthy human population has antibodies against CHIPS that have been shown to interfere with its function in vitro. The aim of this study was to define potential epitopes for human antibodies on the CHIPS surface. We also initiate the process to identify a mutated CHIPS molecule that is not efficiently recognized by preformed anti-CHIPS antibodies and retains anti-inflammatory activity. Results In this paper, we panned peptide displaying phage libraries against a pool of CHIPS specific affinity-purified polyclonal human IgG. The selected peptides could be divided into two groups of sequences. The first group was the most dominant with 36 of the 48 sequenced clones represented. Binding to human affinity-purified IgG was verified by ELISA for a selection of peptide sequences in phage format. For further analysis, one peptide was chemically synthesized and antibodies affinity-purified on this peptide were found to bind the CHIPS molecule as studied by ELISA and Surface Plasmon Resonance. Furthermore, seven potential conformational epitopes responsible for antibody recognition were identified by mapping phage selected peptide sequences on the CHIPS surface as defined in the NMR structure of the recombinant CHIPS31–121 protein. Mapped epitopes were verified by in vitro mutational analysis of the CHIPS molecule. Single mutations introduced in the proposed antibody epitopes were shown to decrease antibody binding to CHIPS. The biological function in terms of C5aR signaling was studied by flow cytometry. A few mutations were shown to affect this biological function as well as the antibody binding. Conclusion Conformational epitopes recognized by human antibodies have been mapped on the CHIPS surface and amino acid residues involved in both antibody and C5aR interaction could be defined. This information has implications for the development of an effective anti-inflammatory agent based on a functional CHIPS molecule with low interaction with human IgG. PMID:19284584
Selective Assembly of Na,K-ATPase α2β2 Heterodimers in the Heart: DISTINCT FUNCTIONAL PROPERTIES AND ISOFORM-SELECTIVE INHIBITORS.

PubMed

Habeck, Michael; Tokhtaeva, Elmira; Nadav, Yotam; Ben Zeev, Efrat; Ferris, Sean P; Kaufman, Randal J; Bab-Dinitz, Elizabeta; Kaplan, Jack H; Dada, Laura A; Farfel, Zvi; Tal, Daniel M; Katz, Adriana; Sachs, George; Vagin, Olga; Karlish, Steven J D

2016-10-28

The Na,K-ATPase α 2 subunit plays a key role in cardiac muscle contraction by regulating intracellular Ca 2+ , whereas α 1 has a more conventional role of maintaining ion homeostasis. The β subunit differentially regulates maturation, trafficking, and activity of α-β heterodimers. It is not known whether the distinct role of α 2 in the heart is related to selective assembly with a particular one of the three β isoforms. We show here by immunofluorescence and co-immunoprecipitation that α 2 is preferentially expressed with β 2 in T-tubules of cardiac myocytes, forming α 2 β 2 heterodimers. We have expressed human α 1 β 1 , α 2 β 1 , α 2 β 2 , and α 2 β 3 in Pichia pastoris, purified the complexes, and compared their functional properties. α 2 β 2 and α 2 β 3 differ significantly from both α 2 β 1 and α 1 β 1 in having a higher K 0.5 K + and lower K 0.5 Na + for activating Na,K-ATPase. These features are the result of a large reduction in binding affinity for extracellular K + and shift of the E 1 P-E 2 P conformational equilibrium toward E 1 P. A screen of perhydro-1,4-oxazepine derivatives of digoxin identified several derivatives (e.g. cyclobutyl) with strongly increased selectivity for inhibition of α 2 β 2 and α 2 β 3 over α 1 β 1 (range 22-33-fold). Molecular modeling suggests a possible basis for isoform selectivity. The preferential assembly, specific T-tubular localization, and low K + affinity of α 2 β 2 could allow an acute response to raised ambient K + concentrations in physiological conditions and explain the importance of α 2 β 2 for cardiac muscle contractility. The high sensitivity of α 2 β 2 to digoxin derivatives explains beneficial effects of cardiac glycosides for treatment of heart failure and potential of α 2 β 2 -selective digoxin derivatives for reducing cardiotoxicity. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.
Electrodrift purification of materials for room temperature radiation detectors

DOEpatents

James, R.B.; Van Scyoc, J.M. III; Schlesinger, T.E.

1997-06-24

A method of purifying nonmetallic, crystalline semiconducting materials useful for room temperature radiation detecting devices by applying an electric field across the material is disclosed. The present invention discloses a simple technology for producing purified ionic semiconducting materials, in particular PbI{sub 2} and preferably HgI{sub 2}, which produces high yields of purified product, requires minimal handling of the material thereby reducing the possibility of introducing or reintroducing impurities into the material, is easy to control, is highly selective for impurities, retains the stoichiometry of the material and employs neither high temperatures nor hazardous materials such as solvents or liquid metals. An electric field is applied to a bulk sample of the material causing impurities present in the sample to drift in a preferred direction. After all of the impurities have been transported to the ends of the sample the current flowing through the sample, a measure of the rate of transport of mobile impurities, falls to a low, steady state value, at which time the end sections of the sample where the impurities have concentrated are removed leaving a bulk sample of higher purity material. Because the method disclosed here only acts on the electrically active impurities, the stoichiometry of the host material remains substantially unaffected. 4 figs.
Electrodrift purification of materials for room temperature radiation detectors

DOEpatents

James, Ralph B.; Van Scyoc, III, John M.; Schlesinger, Tuviah E.

1997-06-24

A method of purifying nonmetallic, crystalline semiconducting materials useful for room temperature radiation detecting devices by applying an electric field across the material. The present invention discloses a simple technology for producing purified ionic semiconducting materials, in particular PbI.sub.2 and preferably HgI.sub.2, which produces high yields of purified product, requires minimal handling of the material thereby reducing the possibility of introducing or reintroducing impurities into the material, is easy to control, is highly selective for impurities, retains the stoichiometry of the material and employs neither high temperatures nor hazardous materials such as solvents or liquid metals. An electric field is applied to a bulk sample of the material causing impurities present in the sample to drift in a preferred direction. After all of the impurities have been transported to the ends of the sample the current flowing through the sample, a measure of the rate of transport of mobile impurities, falls to a low, steady state value, at which time the end sections of the sample where the impurities have concentrated are removed leaving a bulk sample of higher purity material. Because the method disclosed here only acts on the electrically active impurities, the stoichiometry of the host material remains substantially unaffected.
Expression and purification of human and Saccharomyces cerevisiae equilibrative nucleoside transporters.

PubMed

Boswell-Casteel, Rebba C; Johnson, Jennifer M; Roe-Žurž, Zygy; Duggan, Kelli D; Schmitz, Hannah; Hays, Franklin A

2018-02-01

Nucleosides play an essential role in the physiology of eukaryotes by acting as metabolic precursors in de novo nucleic acid synthesis and energy metabolism. Nucleosides also act as ligands for purinergic receptors. Equilibrative nucleoside transporters (ENTs) are polytopic integral membrane proteins that aid in regulating plasmalemmal flux of purine and pyrimidine nucleosides and nucleobases. ENTs exhibit broad substrate selectivity across different isoforms and utilize diverse mechanisms to drive substrate flux across membranes. However, the molecular mechanisms and chemical determinants of ENT-mediated substrate recognition, binding, inhibition, and transport are poorly understood. To determine how ENT-mediated transport occurs at the molecular level, greater chemical insight and assays employing purified protein are essential. This article focuses on the expression and purification of human ENT1, human ENT2, and Saccharomyces cerevisiae ScENT1 using novel expression and purification strategies to isolate recombinant ENTs. ScENT1, hENT1, and hENT2 were expressed in W303 Saccharomyces cerevisiae cells and detergent solubilized from the membrane. After detergent extraction, these ENTs were further purified using immobilized metal affinity chromatography and size exclusion chromatography. This effort resulted in obtaining quantities of purified protein sufficient for future biophysical analysis. Copyright © 2017 Elsevier Inc. All rights reserved.

Purification and characterization of an alginate lyase from marine Bacterium Vibrio sp. mutant strain 510-64.

PubMed

Hu, Xiaoke; Jiang, Xiaolu; Hwang, Huey-Min

2006-08-01

Marine Vibrio sp. 510 was chosen as a parent strain for screening high producers of alginate lyase using the complex mutagenesis of Ethyl Methanesulphonate and UV radiation treatments. The mutant strain Vibrio sp. 510-64 was selected and its alginate lyase activity was increased by 3.87-fold (reaching 46.12 EU/mg) over that of the parent strain. An extracellular alginate lyase was purified from Vibrio sp. 510-64 cultural supernatant by successive fractionation on DEAE Sepharose FF and two steps of Superdex 75. The purified enzyme yielded a single band on SDS-PAGE with the molecular weight of 34.6 kDa. Data of the N-terminal amino acid sequence indicated that this protein might be a novel alginate lyase. The substrate specificity results demonstrated that the alginate lyase had the specificity for poly G block.
Bacterial Reaction Centers Purified with Styrene Maleic Acid Copolymer Retain Native Membrane Functional Properties and Display Enhanced Stability**

PubMed Central

Swainsbury, David J K; Scheidelaar, Stefan; van Grondelle, Rienk; Killian, J Antoinette; Jones, Michael R

2014-01-01

Integral membrane proteins often present daunting challenges for biophysical characterization, a fundamental issue being how to select a surfactant that will optimally preserve the individual structure and functional properties of a given membrane protein. Bacterial reaction centers offer a rare opportunity to compare the properties of an integral membrane protein in different artificial lipid/surfactant environments with those in the native bilayer. Here, we demonstrate that reaction centers purified using a styrene maleic acid copolymer remain associated with a complement of native lipids and do not display the modified functional properties that typically result from detergent solubilization. Direct comparisons show that reaction centers are more stable in this copolymer/lipid environment than in a detergent micelle or even in the native membrane, suggesting a promising new route to exploitation of such photovoltaic integral membrane proteins in device applications. PMID:25212490
The metabolism of neuropeptides. Neurokinin A (substance K) is a substrate for endopeptidase-24.11 but not for peptidyl dipeptidase A (angiotensin-converting enzyme).

PubMed Central

Hooper, N M; Kenny, A J; Turner, A J

1985-01-01

Both endopeptidase-24.11 and peptidyl dipeptidase A have previously been shown to hydrolyse the neuropeptide substance P. The structurally related peptide neurokinin A is also shown to be hydrolysed by pig kidney endopeptidase-24.11. The identified products indicated hydrolysis at two sites, Ser5-Phe6 and Gly8-Leu9, consistent with the known specificity of the enzyme. The pattern of hydrolysis of neurokinin A by synaptic membranes prepared from pig striatum was similar to that observed with purified endopeptidase-24.11, and hydrolysis was substantially abolished by the selective inhibitor phosphoramidon. Peptidyl dipeptidase A purified from pig kidney was shown to hydrolyse substance P but not neurokinin A. It is concluded that endopeptidase-24.11 has the general capacity to hydrolyse and inactivate the family of tachykinin peptides, including substance P and neurokinin A. PMID:2998348
The metabolism of neuropeptides. Neurokinin A (substance K) is a substrate for endopeptidase-24.11 but not for peptidyl dipeptidase A (angiotensin-converting enzyme).

PubMed

Hooper, N M; Kenny, A J; Turner, A J

1985-10-15

Both endopeptidase-24.11 and peptidyl dipeptidase A have previously been shown to hydrolyse the neuropeptide substance P. The structurally related peptide neurokinin A is also shown to be hydrolysed by pig kidney endopeptidase-24.11. The identified products indicated hydrolysis at two sites, Ser5-Phe6 and Gly8-Leu9, consistent with the known specificity of the enzyme. The pattern of hydrolysis of neurokinin A by synaptic membranes prepared from pig striatum was similar to that observed with purified endopeptidase-24.11, and hydrolysis was substantially abolished by the selective inhibitor phosphoramidon. Peptidyl dipeptidase A purified from pig kidney was shown to hydrolyse substance P but not neurokinin A. It is concluded that endopeptidase-24.11 has the general capacity to hydrolyse and inactivate the family of tachykinin peptides, including substance P and neurokinin A.
The Genomic Diversification of the Whole Acinetobacter Genus: Origins, Mechanisms, and Consequences

PubMed Central

Touchon, Marie; Cury, Jean; Yoon, Eun-Jeong; Krizova, Lenka; Cerqueira, Gustavo C.; Murphy, Cheryl; Feldgarden, Michael; Wortman, Jennifer; Clermont, Dominique; Lambert, Thierry; Grillot-Courvalin, Catherine; Nemec, Alexandr; Courvalin, Patrice; Rocha, Eduardo P.C.

2014-01-01

Bacterial genomics has greatly expanded our understanding of microdiversification patterns within a species, but analyses at higher taxonomical levels are necessary to understand and predict the independent rise of pathogens in a genus. We have sampled, sequenced, and assessed the diversity of genomes of validly named and tentative species of the Acinetobacter genus, a clade including major nosocomial pathogens and biotechnologically important species. We inferred a robust global phylogeny and delimited several new putative species. The genus is very ancient and extremely diverse: Genomes of highly divergent species share more orthologs than certain strains within a species. We systematically characterized elements and mechanisms driving genome diversification, such as conjugative elements, insertion sequences, and natural transformation. We found many error-prone polymerases that may play a role in resistance to toxins, antibiotics, and in the generation of genetic variation. Surprisingly, temperate phages, poorly studied in Acinetobacter, were found to account for a significant fraction of most genomes. Accordingly, many genomes encode clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems with some of the largest CRISPR-arrays found so far in bacteria. Integrons are strongly overrepresented in Acinetobacter baumannii, which correlates with its frequent resistance to antibiotics. Our data suggest that A. baumannii arose from an ancient population bottleneck followed by population expansion under strong purifying selection. The outstanding diversification of the species occurred largely by horizontal transfer, including some allelic recombination, at specific hotspots preferentially located close to the replication terminus. Our work sets a quantitative basis to understand the diversification of Acinetobacter into emerging resistant and versatile pathogens. PMID:25313016
Stearoyl-acyl carrier protein desaturases are associated with floral isolation in sexually deceptive orchids

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schluter, P.M.; Shanklin, J.; Xu, S.

The orchids Ophrys sphegodes and O. exaltata are reproductively isolated from each other by the attraction of two different, highly specific pollinator species. For pollinator attraction, flowers chemically mimic the pollinators sex pheromones, the key components of which are alkenes with different double-bond positions. This study identifies genes likely involved in alkene biosynthesis, encoding stearoyl-acyl carrier protein (ACP) desaturase (SAD) homologs. The expression of two isoforms, SAD1 and SAD2, is flower-specific and broadly parallels alkene production during flower development. SAD2 shows a significant association with alkene production, and in vitro assays show that O. sphegodes SAD2 has activity both asmore » an 18:0-ACP {Delta}{sup 9} and a 16:0-ACP {Delta}{sup 4} desaturase. Downstream metabolism of the SAD2 reaction products would give rise to alkenes with double-bonds at position 9 or position 12, matching double-bond positions observed in alkenes in the odor bouquet of O. sphegodes. SAD1 and SAD2 show evidence of purifying selection before, and positive or relaxed purifying selection after gene duplication. By contributing to the production of species-specific alkene bouquets, SAD2 is suggested to contribute to differential pollinator attraction and reproductive isolation among these species. Taken together, these data are consistent with the hypothesis that SAD2 is a florally expressed barrier gene of large phenotypic effect and, possibly, a genic target of pollinator-mediated selection.« less
Stearoyl-acyl carrier protein desaturases are associated with floral isolation in sexually deceptive orchids

PubMed Central

Schlüter, Philipp M.; Xu, Shuqing; Gagliardini, Valeria; Whittle, Edward; Shanklin, John; Grossniklaus, Ueli; Schiestl, Florian P.

2011-01-01

The orchids Ophrys sphegodes and O. exaltata are reproductively isolated from each other by the attraction of two different, highly specific pollinator species. For pollinator attraction, flowers chemically mimic the pollinators’ sex pheromones, the key components of which are alkenes with different double-bond positions. This study identifies genes likely involved in alkene biosynthesis, encoding stearoyl-acyl carrier protein (ACP) desaturase (SAD) homologs. The expression of two isoforms, SAD1 and SAD2, is flower-specific and broadly parallels alkene production during flower development. SAD2 shows a significant association with alkene production, and in vitro assays show that O. sphegodes SAD2 has activity both as an 18:0-ACP Δ9 and a 16:0-ACP Δ4 desaturase. Downstream metabolism of the SAD2 reaction products would give rise to alkenes with double-bonds at position 9 or position 12, matching double-bond positions observed in alkenes in the odor bouquet of O. sphegodes. SAD1 and SAD2 show evidence of purifying selection before, and positive or relaxed purifying selection after gene duplication. By contributing to the production of species-specific alkene bouquets, SAD2 is suggested to contribute to differential pollinator attraction and reproductive isolation among these species. Taken together, these data are consistent with the hypothesis that SAD2 is a florally expressed barrier gene of large phenotypic effect and, possibly, a genic target of pollinator-mediated selection. PMID:21436056
Emergence and Evolution of Hominidae-Specific Coding and Noncoding Genomic Sequences

PubMed Central

Saber, Morteza Mahmoudi; Adeyemi Babarinde, Isaac; Hettiarachchi, Nilmini; Saitou, Naruya

2016-01-01

Family Hominidae, which includes humans and great apes, is recognized for unique complex social behavior and intellectual abilities. Despite the increasing genome data, however, the genomic origin of its phenotypic uniqueness has remained elusive. Clade-specific genes and highly conserved noncoding sequences (HCNSs) are among the high-potential evolutionary candidates involved in driving clade-specific characters and phenotypes. On this premise, we analyzed whole genome sequences along with gene orthology data retrieved from major DNA databases to find Hominidae-specific (HS) genes and HCNSs. We discovered that Down syndrome critical region 4 (DSCR4) is the only experimentally verified gene uniquely present in Hominidae. DSCR4 has no structural homology to any known protein and was inferred to have emerged in several steps through LTR/ERV1, LTR/ERVL retrotransposition, and transversion. Using the genomic distance as neutral evolution threshold, we identified 1,658 HS HCNSs. Polymorphism coverage and derived allele frequency analysis of HS HCNSs showed that these HCNSs are under purifying selection, indicating that they may harbor important functions. They are overrepresented in promoters/untranslated regions, in close proximity of genes involved in sensory perception of sound and developmental process, and also showed a significantly lower nucleosome occupancy probability. Interestingly, many ancestral sequences of the HS HCNSs showed very high evolutionary rates. This suggests that new functions emerged through some kind of positive selection, and then purifying selection started to operate to keep these functions. PMID:27289096
Purification of flavonoids from licorice using an off-line preparative two-dimensional normal-phase liquid chromatography/reversed-phase liquid chromatography method.

PubMed

Fan, Yunpeng; Fu, Yanhui; Fu, Qing; Cai, Jianfeng; Xin, Huaxia; Dai, Mei; Jin, Yu

2016-07-01

An orthogonal (71.9%) off-line preparative two-dimensional normal-phase liquid chromatography/reversed-phase liquid chromatography method coupled with effective sample pretreatment was developed for separation and purification of flavonoids from licorice. Most of the nonflavonoids were firstly removed using a self-made Click TE-Cys (60 μm) solid-phase extraction. In the first dimension, an industrial grade preparative chromatography was employed to purify the crude flavonoids. Click TE-Cys (10 μm) was selected as the stationary phase that provided an excellent separation with high reproducibility. Ethyl acetate/ethanol was selected as the mobile phase owing to their excellent solubility for flavonoids. Flavonoids co-eluted in the first dimension were selected for further purification using reversed-phase liquid chromatography. Multiple compounds could be isolated from one normal-phase fraction and some compounds with bad resolution in one-dimensional liquid chromatography could be prepared in this two-dimensional system owing to the orthogonal separation. Moreover, this two-dimensional liquid chromatography method was beneficial for the preparation of relatively trace flavonoid compounds, which were enriched in the first dimension and further purified in the second dimension. Totally, 24 flavonoid compounds with high purity were obtained. The results demonstrated that the off-line two-dimensional liquid chromatography method was effective for the preparative separation and purification of flavonoids from licorice. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Apolipoprotein L1 confers pH-switchable ion permeability to phospholipid vesicles.

PubMed

Bruno, Jonathan; Pozzi, Nicola; Oliva, Jonathan; Edwards, John C

2017-11-03

Apolipoprotein L1 (ApoL1) is a human serum protein conferring resistance to African trypanosomes, and certain ApoL1 variants increase susceptibility to some progressive kidney diseases. ApoL1 has been hypothesized to function like a pore-forming colicin and has been reported to have permeability effects on both intracellular and plasma membranes. Here, to gain insight into how ApoL1 may function in vivo , we used vesicle-based ion permeability, direct membrane association, and intrinsic fluorescence to study the activities of purified recombinant ApoL1. We found that ApoL1 confers chloride-selective permeability to preformed phospholipid vesicles and that this selectivity is strongly pH-sensitive, with maximal activity at pH 5 and little activity above pH 7. When ApoL1 and lipid were allowed to interact at low pH and were then brought to neutral pH, chloride permeability was suppressed, and potassium permeability was activated. Both chloride and potassium permeability linearly correlated with the mass of ApoL1 in the reaction mixture, and both exhibited lipid selectivity, requiring the presence of negatively charged lipids for activity. Potassium, but not chloride, permease activity required the presence of calcium ions in both the association and activation steps. Direct assessment of ApoL1-lipid associations confirmed that ApoL1 stably associates with phospholipid vesicles, requiring low pH and the presence of negatively charged phospholipids for maximal binding. Intrinsic fluorescence of ApoL1 supported the presence of a significant structural transition when ApoL1 is mixed with lipids at low pH. This pH-switchable ion-selective permeability may explain the different effects of ApoL1 reported in intracellular and plasma membrane environments. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.
Controlled viable release of selectively captured label-free cells in microchannels.

PubMed

Gurkan, Umut Atakan; Anand, Tarini; Tas, Huseyin; Elkan, David; Akay, Altug; Keles, Hasan Onur; Demirci, Utkan

2011-12-07

Selective capture of cells from bodily fluids in microchannels has broadly transformed medicine enabling circulating tumor cell isolation, rapid CD4(+) cell counting for HIV monitoring, and diagnosis of infectious diseases. Although cell capture methods have been demonstrated in microfluidic systems, the release of captured cells remains a significant challenge. Viable retrieval of captured label-free cells in microchannels will enable a new era in biological sciences by allowing cultivation and post-processing. The significant challenge in release comes from the fact that the cells adhere strongly to the microchannel surface, especially when immuno-based immobilization methods are used. Even though fluid shear and enzymes have been used to detach captured cells in microchannels, these methods are known to harm cells and affect cellular characteristics. This paper describes a new technology to release the selectively captured label-free cells in microchannels without the use of fluid shear or enzymes. We have successfully released the captured CD4(+) cells (3.6% of the mononuclear blood cells) from blood in microfluidic channels with high specificity (89% ± 8%), viability (94% ± 4%), and release efficiency (59% ± 4%). We have further validated our system by specifically capturing and controllably releasing the CD34(+) stem cells from whole blood, which were quantified to be 19 cells per million blood cells in the blood samples used in this study. Our results also indicated that both CD4(+) and CD34(+) cells released from the microchannels were healthy and amenable for in vitro culture. Manual flow based microfluidic method utilizes inexpensive, easy to fabricate microchannels allowing selective label-free cell capture and release in less than 10 minutes, which can also be used at the point-of-care. The presented technology can be used to isolate and purify a broad spectrum of cells from mixed populations offering widespread applications in applied biological sciences, such as tissue engineering, regenerative medicine, rare cell and stem cell isolation, proteomic/genomic research, and clonal/population analyses.
Selected HIV-1 Env trimeric formulations act as potent immunogens in a rabbit vaccination model.

PubMed

Heyndrickx, Leo; Stewart-Jones, Guillaume; Jansson, Marianne; Schuitemaker, Hanneke; Bowles, Emma; Buonaguro, Luigi; Grevstad, Berit; Vinner, Lasse; Vereecken, Katleen; Parker, Joe; Ramaswamy, Meghna; Biswas, Priscilla; Vanham, Guido; Scarlatti, Gabriella; Fomsgaard, Anders

2013-01-01

Ten to 30% of HIV-1 infected subjects develop broadly neutralizing antibodies (bNAbs) during chronic infection. We hypothesized that immunizing rabbits with viral envelope glycoproteins (Envs) from these patients may induce bNAbs, when formulated as a trimeric protein and in the presence of an adjuvant. Based on in vitro neutralizing activity in serum, patients with bNAbs were selected for cloning of their HIV-1 Env. Seven stable soluble trimeric gp140 proteins were generated from sequences derived from four adults and two children infected with either clade A or B HIV-1. From one of the clade A Envs both the monomeric and trimeric Env were produced for comparison. Rabbits were immunized with soluble gp120 or trimeric gp140 proteins in combination with the adjuvant dimethyl dioctadecyl ammonium/trehalose dibehenate (CAF01). Env binding in rabbit immune serum was determined using ELISAs based on gp120-IIIB protein. Neutralizing activity of IgG purified from rabbit immune sera was measured with the pseudovirus-TZMbl assay and a PBMC-based neutralization assay for selected experiments. It was initially established that gp140 trimers induce better antibody responses over gp120 monomers and that the adjuvant CAF01 was necessary for such strong responses. Gp140 trimers, based on HIV-1 variants from patients with bNAbs, were able to elicit both gp120IIIB specific IgG and NAbs to Tier 1 viruses of different subtypes. Potency of NAbs closely correlated with titers, and an gp120-binding IgG titer above a threshold of 100,000 was predictive of neutralization capability. Finally, peptide inhibition experiments showed that a large fraction of the neutralizing IgG was directed against the gp120 V3 region. Our results indicate that the strategy of reverse immunology based on selected Env sequences is promising when immunogens are delivered as stabilized trimers in CAF01 adjuvant and that the rabbit is a valuable model for HIV vaccine studies.
Evolution and Expansion of the Prokaryote-Like Lipoxygenase Family in the Brown Alga Saccharina japonica

PubMed Central

Teng, Linhong; Han, Wentao; Fan, Xiao; Xu, Dong; Zhang, Xiaowen; Dittami, Simon M.; Ye, Naihao

2017-01-01

Lipoxygenase (LOX) plays important roles in fatty acid oxidation and lipid mediator biosynthesis. In this study, we give first insights into brown algal LOX evolution. Whole genome searches revealed four, three, and eleven LOXs in Ectocarpus siliculosus, Cladosiphon okamuranus, and Saccharina japonica, respectively. In phylogenetic analyses, LOXs from brown algae form a robust clade with those from prokaryotes, suggesting an ancestral origin and slow evolution. Brown algal LOXs were divided into two clades, C1 and C2 in a phylogenetic tree. Compared to the two species of Ectocarpales, LOX gene expansion occurred in the kelp S. japonica through tandem duplication and segmental duplication. Selection pressure analysis showed that LOX genes in brown algae have undergone strong purifying selection, while the selective constraint in the C2 clade was more relaxed than that in the C1 clade. Furthermore, within each clade, LOXs of S. japonica evolved under more relaxed selection constraints than E. siliculosus and C. okamuranus. Structural modeling showed that unlike LOXs of plants and animals, which contain a β barrel in the N-terminal part of the protein, LOXs in brown algae fold into a single domain. Analysis of previously published transcriptomic data showed that LOXs in E. siliculosus are responsive to hyposaline, hypersaline, oxidative, and copper stresses. Moreover, clear divergence of expression patterns was observed among different life stages, as well as between duplicate gene pairs. In E. siliculosus, all four LOXs are male-biased in immature gametophytes, and mature gametophytes showed significantly higher LOX mRNA levels than immature gametophytes and sporophytes. In S. japonica, however, our RNA-Seq data showed that most LOXs are highly expressed in sporophytes. Even the most recently duplicated gene pairs showed divergent expression patterns, suggesting that functional divergence has likely occurred since LOX genes duplicated, which potentially contributes to the production of various oxylipins in brown algae. PMID:29234336
Flightless I interacts with NMMIIA to promote cell extension formation, which enables collagen remodeling

PubMed Central

Arora, Pamma D.; Wang, Yongqiang; Bresnick, Anne; Janmey, Paul A.; McCulloch, Christopher A.

2015-01-01

We examined the role of the actin-capping protein flightless I (FliI) in collagen remodeling by mouse fibroblasts. FliI-overexpressing cells exhibited reduced spreading on collagen but formed elongated protrusions that stained for myosin10 and fascin and penetrated pores of collagen-coated membranes. Inhibition of Cdc42 blocked formation of cell protrusions. In FliI-knockdown cells, transfection with constitutively active Cdc42 did not enable protrusion formation. FliI-overexpressing cells displayed increased uptake and degradation of exogenous collagen and strongly compacted collagen fibrils, which was blocked by blebbistatin. Mass spectrometry analysis of FliI immunoprecipitates showed that FliI associated with nonmuscle myosin IIA (NMMIIA), which was confirmed by immunoprecipitation. GFP-FliI colocalized with NMMIIA at cell protrusions. Purified FliI containing gelsolin-like domains (GLDs) 1–6 capped actin filaments efficiently, whereas FliI GLD 2–6 did not. Binding assays showed strong interaction of purified FliI protein (GLD 1–6) with the rod domain of NMMIIA (kD = 0.146 μM), whereas FliI GLD 2–6 showed lower binding affinity (kD = 0.8584 μM). Cells expressing FliI GLD 2–6 exhibited fewer cell extensions, did not colocalize with NMMIIA, and showed reduced collagen uptake compared with cells expressing FliI GLD 1–6. We conclude that FliI interacts with NMMIIA to promote cell extension formation, which enables collagen remodeling in fibroblasts. PMID:25877872
Physico-chemical characterization, antioxidant and anticancer activities in vitro of a novel polysaccharide from Melia toosendan Sieb. Et Zucc fruit.

PubMed

He, Liang; Ji, Pengfei; Gong, Xingguo; Li, Weiqi; Cheng, Junwen; Qian, Hua; Song, Xianliang

2011-10-01

A novel water-soluble polysaccharide pMTPS-3, obtained from Melia toosendan Sieb. Et Zucc fruit by hot-water extraction and ethanol precipitation, was fractionated by DEAE-52 cellulose anion-exchange and Sephadex G-100 gel filtration chromatography. Its primary structural features and molecular weight were characterized by Fourier infrared spectrometry (FTIR), gel permeation chromatography (GPC) and gas chromatography (GC). And the antioxidant activities of pMTPS-3 in vitro were evaluated by 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging assay, superoxide radical scavenging assay and hydroxyl radical scavenging assay. The results suggested that pMTPS-3 was a heteropolysaccharide, composed of arabinose, glucose, mannose, and galactose in the molar ratio of 17.3:28.3:41.6:12.6 with molecular weight 26100Da. The purified pMTPS-3 was revealed to have notable scavenging activity against DPPH radical in a concentration-dependent manner and present a moderate inhibition of superoxide radicals with an IC(50) (5.6mg/ml), and potent inhibiting power for hydroxyl radical compared with crude polysaccharide. Further, it exhibited strong inhibition effect in vitro on the growth of human gastric cancer BGC-823 cells. It is strongly evidenced that pMTPS-3 purified from the crude polysaccharides of Melia toosendan Sieb. Et Zucc could be explored as a potential antioxidant and therapeutics. Copyright © 2011 Elsevier B.V. All rights reserved.
Automated Hydrophobic Interaction Chromatography Column Selection for Use in Protein Purification

PubMed Central

Murphy, Patrick J. M.; Stone, Orrin J.; Anderson, Michelle E.

2011-01-01

In contrast to other chromatographic methods for purifying proteins (e.g. gel filtration, affinity, and ion exchange), hydrophobic interaction chromatography (HIC) commonly requires experimental determination (referred to as screening or "scouting") in order to select the most suitable chromatographic medium for purifying a given protein 1. The method presented here describes an automated approach to scouting for an optimal HIC media to be used in protein purification. HIC separates proteins and other biomolecules from a crude lysate based on differences in hydrophobicity. Similar to affinity chromatography (AC) and ion exchange chromatography (IEX), HIC is capable of concentrating the protein of interest as it progresses through the chromatographic process. Proteins best suited for purification by HIC include those with hydrophobic surface regions and able to withstand exposure to salt concentrations in excess of 2 M ammonium sulfate ((NH4)2SO4). HIC is often chosen as a purification method for proteins lacking an affinity tag, and thus unsuitable for AC, and when IEX fails to provide adequate purification. Hydrophobic moieties on the protein surface temporarily bind to a nonpolar ligand coupled to an inert, immobile matrix. The interaction between protein and ligand are highly dependent on the salt concentration of the buffer flowing through the chromatography column, with high ionic concentrations strengthening the protein-ligand interaction and making the protein immobile (i.e. bound inside the column) 2. As salt concentrations decrease, the protein-ligand interaction dissipates, the protein again becomes mobile and elutes from the column. Several HIC media are commercially available in pre-packed columns, each containing one of several hydrophobic ligands (e.g. S-butyl, butyl, octyl, and phenyl) cross-linked at varying densities to agarose beads of a specific diameter 3. Automated column scouting allows for an efficient approach for determining which HIC media should be employed for future, more exhaustive optimization experiments and protein purification runs 4. The specific protein being purified here is recombinant green fluorescent protein (GFP); however, the approach may be adapted for purifying other proteins with one or more hydrophobic surface regions. GFP serves as a useful model protein, due to its stability, unique light absorbance peak at 397 nm, and fluorescence when exposed to UV light 5. Bacterial lysate containing wild type GFP was prepared in a high-salt buffer, loaded into a Bio-Rad DuoFlow medium pressure liquid chromatography system, and adsorbed to HiTrap HIC columns containing different HIC media. The protein was eluted from the columns and analyzed by in-line and post-run detection methods. Buffer blending, dynamic sample loop injection, sequential column selection, multi-wavelength analysis, and split fraction eluate collection increased the functionality of the system and reproducibility of the experimental approach. PMID:21968976
Monooxygenase, a Novel Beta-Cypermethrin Degrading Enzyme from Streptomyces sp

PubMed Central

Xiao, Ying; Deng, Yinyue; Chang, Changqing; Zhong, Guohua; Hu, Meiying; Zhang, Lian-Hui

2013-01-01

The widely used insecticide beta-cypermethrin has become a public concern because of its environmental contamination and toxic effects on mammals. In this study, a novel beta-cypermethrin degrading enzyme designated as CMO was purified to apparent homogeneity from a Streptomyces sp. isolate capable of utilizing beta-cypermethrin as a growth substrate. The native enzyme showed a monomeric structure with a molecular mass of 41 kDa and pI of 5.4. The enzyme exhibited the maximal activity at pH 7.5 and 30°C. It was fairly stable in the pH range from 6.5–8.5 and at temperatures below 10°C. The enzyme activity was significantly stimulated by Fe2+, but strongly inhibited by Ag+, Al3+, and Cu2+. The enzyme catalyzed the degradation of beta-cypermethrin to form five products via hydroxylation and diaryl cleavage. A novel beta-cypermethrin detoxification pathway was proposed based on analysis of these products. The purified enzyme was identified as a monooxygenase by matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry analysis (MALDI-TOF-MS) and N-terminal protein sequencing. Given that all the characterized pyrethroid-degrading enzymes are the members of hydrolase family, CMO represents the first pyrethroid-degrading monooxygenase identified from environmental microorganisms. Taken together, our findings depict a novel pyrethroid degradation mechanism and indicate that the purified enzyme may be a promising candidate for detoxification of beta-cypermethrin and environmental protection. PMID:24098697
Purification and Characterization of Thermostable and Detergent-Stable α-Amylase from Anoxybacillus sp. AH1

PubMed Central

Bekler, Fatma Matpan; Pirinççioğlu, Hemşe; Güven, Reyhan Gül; Güven, Kemal

2016-01-01

Summary A thermostable and detergent-stable α-amylase from a newly isolated Anoxybacillus sp. AH1 was purified and characterized. Maximum enzyme production (1874.8 U/mL) was obtained at 24 h of incubation. The amylase was purified by using Sephadex G-75 gel filtration, after which an 18-fold increase in specific activity and a yield of 9% were achieved. The molecular mass of the purified enzyme was estimated at 85 kDa by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The optimum pH and temperature values of the enzyme were 7.0 and 60 °C, respectively. The enzyme was highly stable in the presence of 30% glycerol, retaining 85% of its original activity at 60 °C within 120 min. Km and vmax values were 0.102 µmol and 0.929 µmol/min, respectively, using Lineweaver-Burk plot. The enzyme activity was increased by various detergents, but it was significantly inhibited in the presence of urea. Mg2+ and Ca2+ also significantly activated α-amylase, while Zn2+, Cu2+ and metal ion chelators ethylenediaminetetraacetic acid (EDTA) and 1,10-phenanthroline (phen) greatly inhibited the enzyme activity. α-Amylase activity was enhanced by β-mercaptoethanol (β-ME) and dithiothreitol (DTT) to a great extent, but inhibited by p-chloromercuribenzoic acid (PCMB). Iodoacetamide (IAA) and N-ethylmaleimide (NEM) had a slight, whereas phenylmethylsulfonyl fluoride (PMSF) had a strong inhibitory effect on the amylase activity. PMID:27904395
A novel serine protease from strawberry (Fragaria ananassa): Purification and biochemical characterization.

PubMed

Alici, Esma Hande; Arabaci, Gulnur

2018-03-27

In this study, a protease enzyme was purified from strawberry by using Sepharose-4B-l-tyrosine-p-amino benzoic acid affinity chromatography. The molecular weight of pure protease was determined 65.8 kDa by SDS-PAGE. The single band observed on the gel showed that the enzyme had a single polypeptide chain and was successfully purified. Purification of the protease by the chromatographic method resulted in a 395.6-fold increase in specific activity (3600 U/mg). Optimum pH and temperature for the enzyme were 6 and 40 °C, respectively. The protease was stable at a wide temperature range of 40 to 70 °C and a pH range of 3.0 to 9.0. Co 2+ ions stimulated protease activity very strongly. Cu 2+ , Hg 2+ , Cd 2+ and Mn 2+ ions significantly inhibited protease activity. While 2-propanol completely inhibited the enzyme, the enzyme maintained its activity better in the presence of ethanol and methanol. The strawberry protease showed the highest specificity towards hemoglobin among all the natural substrates tested. The specificity of the enzyme towards synthetic substrates was also investigated and it was concluded that it has broad substrate specificity. The obtained results indicated that this purified protease was highly-likely a serine protease and its activity was significantly affected by the presence of metal ions. Copyright © 2018. Published by Elsevier B.V.
Non-antigenic and low allergic gelatin produced by specific digestion with an enzyme-coupled matrix.

PubMed

Sakai, Y; Yamato, R; Onuma, M; Kikuta, T; Watanabe, M; Nakayama, T

1998-04-01

Porcine gelatin (heat-denatured collagen) was digested with a bioreactor using an enzyme-coupled matrix (ECM) with purified collagenase. The digested gelatin, FreAlagin type R (M.W. range 200-10000 Da), was further purified by an HPLC system depending upon molecular size. The molecular weight range of the purified fractions, FreAlagin type P and type AD, were 200-500 and 2000-10000 Da, respectively, and glycine was the N-terminal amino acid of both types (> or =93%). ECM has the capability of digesting gelatin at a specific point in the sequence before glycine, and it was determined that FreAlagin type P consists of a tri-peptide fraction with the amino acid sequence Gly-X-Y. No types of FreAlagin exhibited any reactivity with gelatin-specific IgG antibody raised in guinea pigs, and they also possessed an extremely low reactivity with gelatin-specific IgE antibody from the sera of patients who had experienced an anaphylactic reaction against gelatin after vaccination or after eating gelatin-containing foods. From these results, it was determined that FreAlagin types R and AD were non-antigenic, low-allergic gelatins. FreAlagin type R, and especially type AD, had strong adsorption-blocking activity comparable to the level of bovine serum albumin, whereas type P and glycine had virtually no adsorption-blocking activity. Therefore, the new types of gelatin, FreAlagin types R and AD, are suitable for pharmaceutical use to avoid gelatin allergy.

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